AU2017228711B2 - Polynucleotides, Polypeptides Encoded Thereby, and Methods of Using Same for Increasing Abiotic Stress Tolerance and/or Biomass and/or Yield in Plants Expressing Same - Google Patents

Polynucleotides, Polypeptides Encoded Thereby, and Methods of Using Same for Increasing Abiotic Stress Tolerance and/or Biomass and/or Yield in Plants Expressing Same Download PDF

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AU2017228711B2
AU2017228711B2 AU2017228711A AU2017228711A AU2017228711B2 AU 2017228711 B2 AU2017228711 B2 AU 2017228711B2 AU 2017228711 A AU2017228711 A AU 2017228711A AU 2017228711 A AU2017228711 A AU 2017228711A AU 2017228711 B2 AU2017228711 B2 AU 2017228711B2
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plant
growth rate
mab
seed yield
seq
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Sharon Ayal
Alex Diber
Dotan Dimat
Eyal Emmanuel
Michael Gang
Hagai Karchi
Gil Ronen
Judith Basia Vinocur
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Evogene Ltd
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Evogene Ltd
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Priority claimed from AU2014215945A external-priority patent/AU2014215945B2/en
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Abstract

Provided are methods of increasing tolerance of a plant to abiotic stress, and/or increasing biomass, growth rate, vigor and/or yield of a plant. The methods are effected by expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663. Also provided are polynucleotides, nucleic acid constructs, polypeptides and transgenic plants expressing same which can be used to increase tolerance of a plant to abiotic stress, and/or increase biomass, growth rate, vigor and/or yield of a plant.

Description

POLYNUCLEOTIDES, POLYPEPTIDES ENCODED THEREBY, AND METHODS OF USING SAME FOR INCREASING ABIOTIC STRESS TOLERANCE AND/OR BIOMASS AND/OR YIELD IN PLANTS EXPRESSING SAME
FIELD AND BACKGROUND OF THE INVENTION
This application is a divisional of Australian Patent Application No. 2015230753 fded 23 September 2015, which itself was a divisional of Australian Patent Application No. 2014215945 filed 19 August 2014, which itself was a divisional of Australian Patent Application No. 2008278654 filed 24 July 2008, the entire contents of which are herein incorporated by cross[0 reference. The subject matter of this application is related to the applicant’s International Patent Application No. PCT/IL2008/001024 filed 24 July 2008 and claims the benefit of US Provisional Patent Application No. 60/935,046 filed 24 July 2007, the contents of which are incorporated herein by reference in their entirety.
The present invention, in some embodiments thereof, relates to isolated polypeptides and i 5 polynucleotides and more particularly, but not exclusively, to methods of using same for increasing tolerance of a plant to abiotic stress, growth, biomass, vigor and/or yield of a plant.
Abiotic stress (ABS; also referred to as “environmental stress”) conditions such as salinity, drought, flood, suboptimal temperature and toxic chemical pollution, cause substantial damage to agricultural plants. Most plants have evolved strategies to protect themselves against these 10 conditions. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth and yield are profound. Furthermore, most of the crop plants are highly susceptible to ABS and thus necessitate optimal growth conditions for commercial crop yields. Continuous exposure to stress causes major alterations in plant’s metabolism which ultimately leads to cell death and consequently yield loss. Thus, despite extensive research and intensive crop25 protection measures, losses due to abiotic stress conditions remain in the billions of dollars annually.
Drought is a gradual phenomenon, which involves periods of abnormally dry weather that persists long enough to produce serious hydrologic imbalances such as crop damage and water supply shortage. In severe cases, drought can last many years and result in devastating effects on agriculture and water supplies. With burgeoning population and chronic shortage of available fresh 30 water, drought is not only the number one weather-related problem in agriculture, but it also ranks as one of the major natural disasters of all time, causing not only economic damage (e.g., losses from the US drought of 1988 exceeded $40 billion), but also loss of human lives, as in the 1984-1985 drought in the Horn of Africa which led to a famine that killed 750,000 people. Furthermore, drought is associated with increase susceptibility to various diseases.
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For most crop plants, the land regions of the world are too arid. In addition, overuse of available water results in increased loss of agriculturally-usable land (desertification), and increase of salt accumulation in soils adds to the loss of available water in soils.
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Salinity, high salt levels, affects one in five hectares of irrigated land. This condition is only expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops, i.e., wheat, com, rice, potatoes, and soybean, can tolerate excessive salt. Detrimental effects of salt on plants result from both water deficit which leads to osmotic stress (similar to drought stress) and the effect of excess sodium ions on critical biochemical processes. As with freezing and drought, high salt causes water deficit; and the presence of high salt makes it difficult for plant roots to extract water from their environment. Soil salinity is thus one of the more important variables that determine whether a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. Thus, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture, and is worsen by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. On the other hand, germination normally takes place at a salt concentration which is higher than the mean salt level in the whole soil profile.
Germination of many crops is sensitive to temperature. A gene that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates. In addition, seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function. Heat shock may produce a decrease in overall protein synthesis, accompanied by expression of heat shock proteins, e.g., chaperones, which are involved in refolding proteins denatured by heat.
Heat stress often accompanies conditions of low water availability. Heat itself is seen as an interacting stress and adds to the detrimental effects caused by water deficit conditions. Water Evaporative demand exhibits near exponential increases with increases in daytime temperatures and can result in high transpiration rates and low plant water potentials. High-temperature damage to pollen almost always occurs in conjunction with drought stress, and rarely occurs under well-watered conditions.
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Combined stress can alter plant metabolism in novel ways; therefore understanding the interaction between different stresses may be important for the development of strategies to enhance stress tolerance by genetic manipulation.
Excessive chilling conditions, e.g., low, but above freezing, temperatures affect crops of tropical origins, such as soybean, rice, maize, and cotton. Typical chilling damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, pho to inhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. In addition, chilling may lead to yield losses and lower product quality through the delayed ripening of maize.
Water deficit is a common component of many plant stresses. Water deficit occurs in plant cells when the whole plant transpiration rate exceeds the water uptake. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration.
Salt and drought stress signal transduction consist of ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as S0S1. The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or cold stress responses.
Common aspects of drought, cold and salt stress response [Reviewed in Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139] include: (a) transient changes in the cytoplasmic calcium levels early in the signaling event [Knight, (2000) Int. Rev. Cytol. 195: 269-324; Sanders et al. (1999) Plant Cell 11: 691-706]; (b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs) and protein phosphatases [Merlot et al. (2001) Plant J. 25: 295-303; Tahtiharju and Palva (2001) Plant J. 26: 461-470]; (c) increases in abscisic acid levels in response to stress triggering a subset of responses; (d) inositol phosphates as signal molecules (at least for a subset of the stress responsive transcriptional changes [Xiong et al. (2001) Genes Dev. 15: 19711984]; (e) activation of phospholipases which in turn generates a diverse array of second messenger molecules, some of which might regulate the activity of stress responsive
2017228711 15 Sep 2017 kinases [e.g., phospholipase D; Frank et al. (2000) Plant Cell 12: 111-124]; (f) induction of late embryogenesis abundant (LEA) type genes including the CRT/DRE responsive COR/RD genes; (g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars [Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol. 51: 463-499)]; and (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals.
Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.
Several genes which increase tolerance to cold or salt stress can also improve drought stress protection, these include for example, the transcription factor AtCBF/DREBl, OsCDPK7 (Saijo et al. 2000, Plant J. 23: 319-327) or AVP1 (a vacuolar pyrophosphatase-proton pump, Gaxiola et al. 2001, Proc. Natl. Acad. Sei. USA 98: 11444-11449).
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies used to develop new lines of plants that exhibit tolerance to ABS are relatively inefficient since they are tedious, time consuming and of unpredictable outcome. Furthermore, limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to ABS tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways.
Genetic engineering efforts, aimed at conferring abiotic stress tolerance to transgenic crops, have been described in the art. Studies by Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002), Quesada et al. (Plant Physiol. 130:951-963, 2002), Holmstrom et al. (Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257, 1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) and Tarczynski et al. (Science 259: 508-510, 1993) have all attempted at generating stress tolerant plants.
In addition, several U.S. patents and patent applications also describe polynucleotides associated with stress tolerance and their use in generating stress
2017228711 15 Sep 2017 tolerant plants. U.S. Pat. Nos. 5,296,462 and 5,356,816 describe transforming plants with polynucleotides encoding proteins involved in cold adaptation in Arabidopsis thaliana for promoting cold tolerance.
U.S. Pat. No. 6,670,528 describes transforming plants with polynucleotides encoding polypeptides binding to stress responsive elements for promoting tolerance to abiotic stress.
U.S. Pat. No. 6,720,477 describes transforming plants with a polynucleotide encoding a signal transduction stress-related protein, capable of increasing tolerance of the transformed plants to abiotic stress.
U.S. Application Ser. Nos. 09/938842 and 10/342224 describe abiotic stressrelated genes and their use to confer upon plants tolerance to abiotic stress.
U.S. Application Ser. No. 10/231035 describes overexpressing a molybdenum cofactor sulfurase in plants for increasing tolerance to abiotic stress.
W02004/104162 to Evogene Ltd. teaches polynucleotide sequences and methods of utilizing same for increasing the tolerance of a plant to abiotic stresses and/or increasing the biomass of a plant.
W02007/020638 to Evogene Ltd. teaches polynucleotide sequences and methods of utilizing same for increasing the tolerance of a plant to abiotic stresses and/or increasing the biomass, vigor and/or yield of a plant.
W02007/049275 to Evogene Ltd. teaches isolated polypeptides, polynucleotides encoding same for increasing tolerance of a plant to abiotic stress, and/or for increasing biomass, vigor and/or yield of a plant.
Additional background art includes U.S. Patent Appl. Nos. 20060183137A1 Al and 20030056249A1.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of increasing tolerance of a plant to abiotic stress, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202206, 208-211, 213-391, 1655, 961-1529, and 1660-1663, thereby increasing the tolerance of the plant to abiotic stress.
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According to an aspect of some embodiments of the present invention there is provided a method of increasing tolerance of a plant to abiotic stress, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 16601663, thereby increasing the tolerance of the plant to abiotic stress.
According to an aspect of some embodiments of the present invention there is provided a method of increasing biomass, growth rate, vigor and/or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202206, 208-211, 213-391, 1655, 961-1529, and 1660-1663, thereby increasing the biomass, growth rate, vigor and/or yield of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of increasing biomass, growth rate, vigor and/or yield of a plant, the method comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 16601663, thereby increasing the biomass, growth rate, vigor and/or yield of the plant.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence at least 90 % identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540,
1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538,1549,
1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548,1556,
1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667,1542,
1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
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According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the isolated polynucleotide and a promoter for directing transcription of the nucleic acid sequence.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide, comprising an amino acid sequence at least 90 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 9611529, and 1660-1663.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising an exogenous polypeptide comprising an amino acid sequence at least 90 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising an exogenous polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising an exogenous polynucleotide comprising a nucleic acid sequence at least 90 % homologous to the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising an exogenous polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
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According to some embodiments of the invention, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547,
1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
According to some embodiments of the invention, the polynucleotide is selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533, 1538,
1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
According to some embodiments of the invention, the amino acid sequence is selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to some embodiments of the invention, the polypeptide is selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to some embodiments of the invention, the plant cell forms a part of a plant.
According to some embodiments of the invention, the abiotic stress is selected from the group consisting of salinity, drought, water deprivation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.
According to some embodiments of the invention, the method further comprising growing the plant expressing the exogenous polynucleotide under the abiotic stress.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
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Definitions of the specific embodiments of the invention as claimed herein follow.
According to a first embodiment of the invention, there is provided a method of increasing biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance of a plant, the method comprising over-expressing within the plant a polypeptide 5 comprising an amino acid sequence at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 271, wherein when said abiotic stress is salinity stress then said tolerance to said salinity stress is an increase in said seed yield and/or an increase in said growth rate under salinity stress as compared to a native plant of the same species which is grown under the same growth LO conditions, thereby increasing the biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance of the plant, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency.
According to a second embodiment of the invention, there is provided a method of L5 growing a crop comprising growing a crop plant over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, wherein said crop plant is derived from parent plants over-expressing said polypeptide as compared to a native plant of the same species which is grown under the same growth conditions, and which have been selected for increased biomass, growth rate, seed yield, >0 nitrogen use efficiency and/or abiotic stress tolerance of a plant, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency, and said crop plant over-expressing said polypeptide having said increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, wherein said tolerance to said salinity stress is an increase in said seed yield and/or an increase in said growth rate under said salinity 25 stress as compared to a native plant of the same species which is grown under the same growth conditions, thereby growing the crop.
According to a third embodiment of the invention, there is provided a method of selecting a plant having increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, the method comprising:
(a) providing plants over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, (b) selecting said plants of step (a) for increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency, wherein said tolerance to
8b
2017228711 11 Dec 2018 said salinity stress is an increase in said seed yield and/or an increase in said growth rate under salinity stress as compared to a native plant of the same species which is grown under the same growth conditions, and (c) growing a crop of said plant selected in step (b), thereby selecting the plant having the increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance.
According to a fourth embodiment of the invention, there is provided a method of increasing root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or increased seed yield of a plant as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of a control plant of the same species which is grown under the same growth conditions, comprising:
(a) over-expressing within the plant a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, and (b) selecting from plants resultant of step (a) a plant exhibiting an increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said selecting is for a plant exhibiting said increased seed yield and/or said increased growth rate as compared to said control plant of the same species which is grown under the same growth conditions, thereby increasing the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of the plant as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of the control 10 plant of the same species which is grown under the same growth conditions.
According to a fifth embodiment of the invention, there is provided a method of producing a crop comprising growing a crop plant over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, wherein said crop plant is derived from parent plants selected for increased root length, root 15 coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said parent plants are selected for said increased seed yield and/or said increased growth rate as 20 compared to said control plant of the same species which is grown under the same growth
8c
2017228711 11 Dec 2018 conditions, and said crop plant having said increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield, thereby producing the crop.
According to a sixth embodiment of the invention, there is provided a method of selecting a plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, the method comprising:
(a) providing plants transformed with a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide, wherein said polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 271, and a heterologous promoter for directing transcription of said nucleic acid sequence in a plant cell, and;
(b) selecting from said plants a plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said selecting is for a plant having said increased seed yield and/or said increased growth rate as compared to said control plant of the same species which is grown under the same growth conditions, thereby selecting the plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to the control plant 5 of the same species which is grown under the same growth conditions.
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BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIG. 1 is a schematic illustration of the pGI binary plasmid used for expressing the isolated polynucleotide sequences of the invention. RB - T-DNA right border; LB T-DNA left border; H- Hind\\\ restriction enzyme; X - Xbal restriction enzyme; B BamHI restriction enzyme; S - Sall restriction enzyme; Sm - Smal restriction enzyme; R-I - EcoRI restriction enzyme; Sc - SacI/SstI/Ecll36II; (numbers) - Length in basepairs; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poly-A signal (polyadenylation signal); GUSintron - the GUS reporter gene (coding sequence and intron) The isolated polynucleotide sequences of the invention were cloned into the vector while replacing the GUSintron reporter gene.
FIGs. 2a-b are images depicting visualization of root development of plants grown in transparent agar plates. The different transgenes were grown in transparent agar plates for 17 days and the plates were photographed every 2 days starting at day 7. Figure 2a - An image of a photograph of plants taken following 12 days on agar plates. Figure 2b - An image of root analysis in which the length of the root measured is represented by a red arrow.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to isolated polypeptides and polynucleotides encoding same, and more particularly, but not exclusively, to methods of using same for increasing tolerance to abiotic stress, growth rate, yield, biomass and/or vigor of a plant.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set
2017228711 15 Sep 2017 forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
While reducing the invention to practice, the present inventors have identified novel polypeptides and polynucleotides which can be used to increase tolerance to abiotic stress, and improve growth rate, biomass, yield and/or vigor of a plant.
Thus, as shown in the Examples section which follows, the present inventors have employed a bioinformatics approach which combines clustering and assembly of sequences from databases of the Arabidopsis, rice and other publicly available plant genomes, expressed sequence tags (ESTs), protein and pathway databases and QTL information with a digital expression profile (“electronic Northern Blot”) and identified polynucleotides and polypeptides which can increase tolerance to abiotic stress, and improve growth, biomass, yield and vigor (SEQ ID NOs: 1-200 and 1653 for polynucleotides; SEQ ID NOs:201-391 and 1655 for polypeptides; Table 1, Example 1). Putative ABST orthologs from monocot species were identified by alignments of ortholog sequences and digital expression profiles (SEQ ID NOs:392-960, 1656-1659 for polynucleotides; SEQ ID NOs:961-1529, 1660-1663 for polypeptides; Table 2, Example 1). As is further described in Tables 3 and 4 of the Examples section which follows, representative polynucleotides were cloned (polynucleotide SEQ ID NOs: 1530, 1538, 1532, 1549, 1665, 1566, 1554, 1563, 1557, 1561, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543 and 1668). Additional polynucleotides having optimized nucleic acid sequences were prepared (polynucleotide SEQ ID NOs: 1531, 1539, 1533, 1550, 1558, 1562, 1565, 1541, 1667, 1542, 1544, 1537, 1551 and 1545). As is further described in the Examples section which follows, transgenic plants exogenously expressing the cloned and/or optimized polynucleotides of the invention were generated. As shown in Tables 5-76, these plants exhibit increased seedling weight, root coverage, root length, and relative growth rate when grown under osmotic stress (in the presence of 25 % PEG), nitrogen deficiency (in the presence of 0.75 mM Nitrogen) or regular conditions. In addition, as shown in Tables 77-188, plants exogenously expressing the polynucleotides of the invention exhibit increased rosette area, rosette diameter, leaf average area, relative growth rate of the above, plants biomass, plant seed yield, 1000 seed weight, and harvest index when grown under salinity stress or normal conditions. Altogether, these results suggest the use of the
2017228711 15 Sep 2017 novel polynucleotides and polypeptides of the invention for increasing abiotic stress tolerance, and improving growth rate biomass, vigor and/or yield of a plant.
Thus, according to one aspect of the invention, there is provided a method of increasing abiotic stress tolerance, growth rate, biomass, yield and/or vigor of a plant. The method is effected by expressing within the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 60 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
The phrase abiotic stress as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, water deprivation, water deficit, drought, flooding, freezing, low or high temperature (e.g., chilling or excessive heat), toxic chemical pollution, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution or UV irradiation. The implications of abiotic stress are discussed in the Background section.
The phrase “abiotic stress tolerance” as used herein refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
As used herein the phrase plant biomass refers to the amount (measured in grams of air-dry or dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area.
As used herein the phrase plant yield refers to the amount (as determined by weight, volume or size) or quantity (numbers) of tissue produced or harvested per plant or per growing season. Hence increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
As used herein the phrase plant vigor refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increase vigor could determine or affect the plant yield or the yield per growing time or growing area.
As used herein the term increasing refers to at least about 2 %, at least about 3 %, at least about 4 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 % or greater increase in plant abiotic stress tolerance, growth, biomass, yield and/or vigor as compared to a native plant [i.e., a plant
2017228711 15 Sep 2017 not modified with the biomolecules (polynucleotide or polypeptides) of the invention, e.g., a non-transformed plant of the same species which is grown under the same growth conditions).
As used herein, the phrase exogenous polynucleotide refers to a heterologous nucleic acid sequence which may not be naturally expressed within the plant or which overexpression in the plant is desired. The exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the plant.
As mentioned, the exogenous polynucleotide of the invention encodes a polypeptide having an amino acid sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 81 %, at least about 82 %, at least about 83 %, at least about 84 %, at least about 85 %, at least about 86 %, at least about 87 %, at least about 88 %, at least about 89 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or more say 100 % homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:201, 207, 212, 202-206, 208-211, 213-391, 1655, 9611529, and 1660-1663.
Homology (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastP or TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the sixframe conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
Homologous sequences include both orthologous and paralogous sequences. The term “paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes. The term “orthologous” relates to homologous genes in different organisms due to ancestral relationship.
One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the
2017228711 15 Sep 2017 sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov. If orthologues in rice were sought, the sequence-ofinterest would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. The blast results may be filtered. The fulllength sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-ofinterest is derived. The results of the first and second blasts are then compared. An orthologue is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-ofinterest) as the best hit. Using the same rational a paralogue (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used [Hypertext Transfer Protocol://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor-joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth by SEQ ID NO:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, 1660-1662 or 1663.
According to some embodiments of the invention the exogenous polynucleotide comprises a nucleic acid sequence which is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 81 %, at least about 82 %, at least about 83 %, at least about 84 %, at least about 85 %, at least about 86 %, at least about 87 %, at least about 88 %, at least about 89 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, e.g., 100 % identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1530, 1561, 1532, 1531, 1562, 1533,1538,
1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547,1548,
1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541,1667,
1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1659.
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Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.
According to some embodiments of the invention the exogenous polynucleotide is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 81 %, at least about 82 %, at least about 83 %, at least about 84 %, at least about 85 %, at least about 86 %, at least about 87 %, at least about 88 %, at least about 89 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, e.g., 100 % identical to the polynucleotide selected from the group consisting of SEQ ID NOs:1530,
1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534,
1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668,
1539, 1550, 1558, 1565,1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392960, and 1656-1659.
According to some embodiments of the invention the exogenous polynucleotide is set forth by SEQ IDNO:1530, 1561, 1532, 1531, 1562, 1533, 1538, 1549, 1665, 1566, 1554, 1563, 1557, 1564, 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543, 1668, 1539, 1550, 1558, 1565, 1541, 1667, 1542, 1544, 1537, 1551, 1545, 1-200, 1653, 392-960, and 1656-1658 or 1659.
As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As used herein the phrase complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
As used herein the phrase genomic polynucleotide sequence refers to a sequence derived (identified or isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase composite polynucleotide sequence refers to a sequence, which is at least partially complementary and at least partially genomic. A
2017228711 15 Sep 2017 composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
Nucleic acid sequences encoding the polypeptides of the present invention may be optimized for expression. A non-limiting example of an optimized nucleic acid sequence is provided in SEQ ID NO: 1531, which encodes the polypeptide comprising the amino acid sequence set forth by SEQ ID NO:201. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
The phrase codon optimization refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU = n = 1 N [ ( Xn - Yn)/Yn] 2/N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A Table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization Tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of
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Agrobiological Sciences) DNA bank in Japan (Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/). The Codon Usage Database contains codon usage Tables for a number of different species, with each codon usage Table having been statistically determined based on the data present in Genbank.
By using the above Tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturallyoccurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more lessfavored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statisticallyfavored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
Thus, the invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
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The invention provides an isolated polypeptide having an amino acid sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 81 %, at least about 82 %, at least about 83 %, at least about 84 %, at least about 85 %, at least about 86 %, at least about 87 %, at least about 88 %, at least about 89 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or more say 100 % homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1663.
According to some embodiments of the invention, the polypeptide is set forth by SEQ ID NO:201, 207, 212, 202-206, 208-211, 213-391, 1655, 961-1529, and 1660-1662 or 1663.
The invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man induced, either randomly or in a targeted fashion.
The term 'plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia
2017228711 15 Sep 2017 squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.
According to some embodiments of the invention, the plant used by the method of the invention is a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, popular and cotton.
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Expressing the exogenous polynucleotide of the invention within the plant can be effected by transforming one or more cells of the plant with the exogenous polynucleotide, followed by generating a mature plant from the transformed cells and cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
According to some embodiments of the invention, the transformation is effected by introducing to the plant cell a nucleic acid construct which includes the exogenous polynucleotide of some embodiments of the invention and at least one promoter capable of directing transcription of the exogenous polynucleotide in the plant cell. Further details of suitable transformation approaches are provided hereinbelow.
As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls where (e.g., which portion of a plant) and/or when (e.g., at which stage or condition in the lifetime of an organism) the gene is expressed.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter, a tissue-specific, or an abiotic stress-inducible promoter.
Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO:1546; Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO:1652; see PCT Publication No. W004081173A2); maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant I Nov;2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675689, 1992); Rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant I. 10( 1); 107-121, 1996), constitutive root tip CT2 promoter (SEQ ID NO:1535; see also PCT application No. IL/2005/000627) and Synthetic Super MAS (Ni et al., The Plant lournal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5.608,144; 5,604,121; 5.569,597: 5.466,785; 5,399,680; 5,268,463; and 5,608,142.
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Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sei. USA 90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235- 245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al., Plant Mol Biol, 143).323-32 1990), napA (Stalberg, et al., Planta 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171- 184, 1997), sunflower oleosin (Cummins, etal., Plant Mol. Biol. 19: 873876, 1992)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMBO3:1409-15, 1984), Barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996), Barley DOF (Mena et al., The Plant Journal, 116(1): 53- 62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice -globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885- 889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma- kafirin (PMB 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSHI (Sato et al., Proc. Nati. Acad. Sei. USA, 93: 8117-8122), KNOX (Postma-Haarsma ef al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245; 1989), apetala- 3].
Suitable abiotic stress-inducible promoters include, but not limited to, saltinducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rabl7 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266, 1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol.
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Biol. 39:373-380, 1999); heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).
The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication. According to some embodiments of the invention, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
The nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276).
The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant
2017228711 15 Sep 2017 cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In micro injection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced
2017228711 15 Sep 2017 such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. For this reason it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.
Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
According to some embodiments of the invention, the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.
Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV),
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EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.
According to some embodiments of the invention, the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tailor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
Construction of plant RNA viruses for the introduction and expression of nonviral exogenous polynucleotide sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990) 269:73-76; and U.S. Pat. No. 5,316,931.
When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the
2017228711 15 Sep 2017 viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The
RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral
RNA.
In one embodiment, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a nonnative plant viral subgenomic promoters if more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
In a second embodiment, a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a nonnative coat protein coding sequence.
In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the
2017228711 15 Sep 2017 sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
In a fourth embodiment, a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
Techniques for inoculation of viruses to plants may be found in Foster and Taylor, eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S.A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D.G.A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.
In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
A technique for introducing exogenous polynucleotide sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotide is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast
2017228711 15 Sep 2017 genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
Since abiotic stress tolerance, growth, biomass, yield and/or vigor in plants can involve multiple genes acting additively or in synergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002), the present invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior effect on abiotic stress tolerance, growth, biomass, yield and/or vigor.
Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences. To enable co-translation of the different polypeptides encoded by the polycistronic messager RNA, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5' end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. Alternatively, the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
The plant cell transformed with the construct including a plurality of different exogenous polynucleotides can be regenerated into a mature plant, using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides can be effected by introducing different nucleic acid constructs, including different exogenous
2017228711 15 Sep 2017 polynucleotides, into a plurality of plants. The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior abiotic stress tolerance, growth, biomass, yield and/or vigor traits, using conventional plant breeding techniques.
According to some embodiments of the invention, the plant expressing the exogenous polynucleotide(s) is grown under normal conditions.
According to some embodiments of the invention, the method further comprising growing the plant expressing the exogenous polynucleotide(s) under the abiotic stress.
Thus, the invention encompasses plants exogenously expressing (as described above) the polynucleotide(s) and/or polypeptide(s) of the invention. Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Enzyme-Linked ImmunoSorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.
Methods of determining the level in the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-m situ hybridization.
The polynucleotides and polypeptides described hereinabove can be used in a wide range of economical plants, in a safe and cost effective manner.
The effect of the transgene (the exogenous polynucleotide encoding the polypeptide) on abiotic stress tolerance, growth, biomass, yield and/or vigor can be determined using known methods.
Abiotic stress tolerance - Transformed (i.e., expressing the transgene) and nontransformed (wild type) plants are exposed to an abiotic stress condition, such as water deprivation, suboptimal temperature (low temperature, high temperature), nutrient deficiency, nutrient excess, a salt stress condition, osmotic stress, heavy metal toxicity, anaerobiosis, atmospheric pollution and UV irradiation.
Salinity tolerance assay — Transgenic plants with tolerance to high salt concentrations are expected to exhibit better germination, seedling vigor or growth in high salt. Salt stress can be effected in many ways such as, for example, by irrigating
2017228711 15 Sep 2017 the plants with a hyperosmotic solution, by cultivating the plants hydroponically in a hyperosmotic growth solution (e.g., Hoagland solution with added salt), or by culturing the plants in a hyperosmotic growth medium [e.g., 50 % Murashige-Skoog medium (MS medium) with added salt]. Since different plants vary considerably in their tolerance to salinity, the salt concentration in the irrigation water, growth solution, or growth medium can be adjusted according to the specific characteristics of the specific plant cultivar or variety, so as to inflict a mild or moderate effect on the physiology and/or morphology of the plants (for guidelines as to appropriate concentration see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and reference therein).
For example, a salinity tolerance test can be performed by irrigating plants at different developmental stages with increasing concentrations of sodium chloride (for example 50 mM, 100 mM, 200 mM, 400 mM NaCl) applied from the bottom and from above to ensure even dispersal of salt. Following exposure to the stress condition the plants are frequently monitored until substantial physiological and/or morphological effects appear in wild type plants. Thus, the external phenotypic appearance, degree of wilting and overall success to reach maturity and yield progeny are compared between control and transgenic plants. Quantitative parameters of tolerance measured include, but are not limited to, the average wet and dry weight, growth rate, leaf size, leaf coverage (overall leaf area), the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as abiotic stress tolerant plants.
Osmotic tolerance test - Osmotic stress assays (including sodium chloride and PEG assays) are conducted to determine if an osmotic stress phenotype was sodium chloride-specific or if it was a general osmotic stress related phenotype. Plants which are tolerant to osmotic stress may have more tolerance to drought and/or freezing. For salt and osmotic stress experiments, the medium is supplemented for example with 50 mM, 100 mM, 200 mM NaCl or 15 %, 20 % or 25 % PEG. See also Examples 6 and 7 of the Examples section which follows.
Drought tolerance assay/Osmoticum assay - Tolerance to drought is performed to identify the genes conferring better plant survival after acute water deprivation. To
2017228711 15 Sep 2017 analyze whether the transgenic plants are more tolerant to drought, an osmotic stress produced by the non-ionic osmolyte sorbitol in the medium can be performed. Control and transgenic plants are germinated and grown in plant-agar plates for 4 days, after which they are transferred to plates containing 500 mM sorbitol. The treatment causes growth retardation, then both control and transgenic plants are compared, by measuring plant weight (wet and dry), yield, and by growth rates measured as time to flowering.
Conversely, soil-based drought screens are performed with plants overexpressing the polynucleotides detailed above. Seeds from control Arabidopsis plants, or other transgenic plants overexpressing the polypeptide of the invention are germinated and transferred to pots. Drought stress is obtained after irrigation is ceased accompanied by placing the pots on absorbent paper to enhance the soil-drying rate. Transgenic and control plants are compared to each other when the majority of the control plants develop severe wilting. Plants are re-watered after obtaining a significant fraction of the control plants displaying a severe wilting. Plants are ranked comparing to controls for each of two criteria: tolerance to the drought conditions and recovery (survival) following re-watering.
Cold stress tolerance - One way to analyze cold stress is as follows. Mature (25 day old) plants are transferred to 4 °C chambers for 1 or 2 weeks, with constitutive light. Later on plants are moved back to greenhouse. Two weeks later damages from chilling period, resulting in growth retardation and other phenotypes, are compared between control and transgenic plants, by measuring plant weight (wet and dry), and by comparing growth rates measured as time to flowering, plant size, yield, and the like.
Heat stress tolerance - One way to measure heat stress tolerance is by exposing the plants to temperatures above 34 °C for a certain period. Plant tolerance is examined after transferring the plants back to 22 °C for recovery and evaluation after 5 days relative to internal controls (non-transgenic plants) or plants not exposed to neither cold or heat stress.
Germination tests - Germination tests compare the percentage of seeds from transgenic plants that could complete the germination process to the percentage of seeds from control plants that are treated in the same manner. Normal conditions are considered for example, incubations at 22 °C under 22-hour light 2-hour dark daily cycles. Evaluation of germination and seedling vigor is conducted between 4 and 14
2017228711 15 Sep 2017 days after planting. The basal media is 50 % MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
Germination is checked also at unfavorable conditions such as cold (incubating at temperatures lower than 10 °C instead of 22 °C) or using seed inhibition solutions that contain high concentrations of an osmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrations of salt (of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).
Effect of the transgene on plant’s growth, biomass, yield and/or vigor - Plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight and the like per time.
The growth rate can be measured using digital analysis of growing plants. For example, images of plants growing in greenhouse on plot basis can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference of rosette area between days of sampling divided by the difference in days between samples.
Measurements of seed yield can be done by collecting the total seeds from 8-16 plants together, weighting them using analytical balance and dividing the total weight by the number of plants. Seed per growing area can be calculated in the same manner while taking into account the growing area given to a single plant. Increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants capable of growing in a given area.
Evaluation of the seed yield per plant can be done by measuring the amount (weight or size) or quantity (i.e., number) of dry seeds produced and harvested from 816 plants and divided by the number of plants.
Evaluation of growth rate can be done by measuring plant biomass produced, rosette area, leaf size or root length per time (can be measured in cm per day of leaf area).
Fiber length can be measured using fibrograph. The fibrograph system was used to compute length in terms of Upper Half Mean length. The upper half mean (UHM) is the average length of longer half of the fiber distribution. The fibrograph measures length in span lengths at a given percentage point (Hypertext Transfer Protocol://World Wide Web (dot) cottoninc (dot) com/ClassificationofCotton/?Pg=4#Length).
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Thus, the present invention is of high agricultural value for promoting the yield of commercially desired crops (e.g., biomass of vegetative organ such as poplar wood, or reproductive organ such as number of seeds or seed biomass).
As used herein the term “about” refers to ± 10 %.
The terms comprises, comprising, includes, including, “having” and their conjugates mean including but not limited to.
The term “consisting of means “including and limited to”.
The term consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form a, an and the include plural references unless the context clearly dictates otherwise. For example, the term a compound or at least one compound may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known
2017228711 15 Sep 2017 manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, Molecular Cloning: A laboratory Manual Sambrook et al., (1989); Current Protocols in Molecular Biology Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; Cell Biology: A Laboratory Handbook, Volumes I-III Cellis, J. E., ed. (1994); Current Protocols in Immunology Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932;
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3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; Oligonucleotide Synthesis Gait, M. J., ed. (1984); “Nucleic Acid Hybridization Hames, B. D., and Higgins S. J., eds. (1985); Transcription and Translation Hames, B. D., and Higgins S. J., Eds. (1984); Animal Cell Culture Freshney, R. I., ed. (1986); Immobilized Cells and Enzymes IRL Press, (1986); A Practical Guide to Molecular Cloning Perbal, B., (1984) and Methods in Enzymology Vol. 1-317, Academic Press; PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990); Marshak et al., Strategies for Protein Purification and Characterization - A Laboratory Course Manual CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
EXAMPLE 1
IDENTIFYING PUTATIVE ABIOTIC STRESS- TOLERANCE AND OR YIELD/BIOMASS INCREASE GENES
The present inventors have identified genes which increase abiotic stresstolerance (ABST) and/or growth rate/yield/biomass/vigor, as follows. The genes were validated in vivo as previously described in W02004/104162 to the present assignee. All nucleotide sequence datasets used here were originated from publicly available databases. Sequence data from 50 different species (mainly plant species) was introduced into a single, comprehensive database. Other information on gene expression, protein annotation, enzymes and pathways were also incorporated. Major databases used include:
• Genomes o Arabidopsis genome [TAIR genome version 6 (Hypertext Transfer
Protocol://World Wide Web (dot) arabidopsis (dot) org/)] o Rice genome [IRGSP build 4.0 (Hypertext Transfer Protocol://rgp (dot) dna (dot) affrc (dot) go (dot) jp/IRGSP/)].
2017228711 15 Sep 2017 o Poplar [Populus trichocarpa release 1.1 from JGI (assembly release vl .0) (Hypertext Transfer Protocol://World Wide Web (dot) genome (dot) jgi-psf (dot) org/)] o Brachypodium [JGI 4x assembly Hypertext Transfer Protocol://World Wide Web (dot) brachpodium (dot) org)] o Soybean [DOE-JGI SCP, version GlymaO (Hypertext Transfer Protocol://World Wide Web (dot) phytozome (dot) net/)] o Grape [NCBI WGS assembly ftp://ftp (dot) ncbi (dot) nih (dot) gov/ genbank/wgs/)] o Castobean [TIGR/J Craig Venter Institute 4x assemby o Hypertext Transfer Protocol://msc (dot) jcvi (dot) org/r_communis o Sorghum [DOE-JGI SCP, version Sbil Hypertext Transfer Protocol://World Wide Web (dot) phytozome (dot) net/)].
• Expressed EST and mRNA sequences were extracted from o GeneBank versions 154, 157, 160, 161, 164, and 165 (Hypertext Transfer
Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/dbEST/) o RefSeq (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/RcfScq/).
o TAIR (Hypertext Transfer Protocol ://World Wide Web (dot) arabidopsis (dot) org/).
• Protein and pathway databases o Uniprot (Hypertext Transfer Protocol ://World Wide
W eb .expasy.uniprot. org/).
o AraCyc (Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/biocyc/index (dot) jsp).
o ENZYME (Hypertext Transfer Protocol://expasy.org/enzyme/).
• Microarray datasets were downloaded from o GEO (Hypertext Transfer Protocol://World Wide
Web.ncbi.nlm.nih.gov/geo/) o TAIR (Hypertext Transfer Protocol://World Wide Web.arabidopsis.org/).
o Proprietary Evogene's cotton fiber microarray data
2017228711 15 Sep 2017 • QTL information o Gramene (Hypertext Transfer Protocol ://World Wide Web (dot) gramene (dot) org/qtl/).
Database Assembly was performed to build a wide, rich, reliable annotated and easy to analyze database comprised of publicly available genomic mRNA, ESTs DNA sequences, data from various crops as well as gene expression, protein annotation and pathway data QTLs, and other relevant information.
Database assembly is comprised of a toolbox of gene refining, structuring, annotation and analysis tools enabling to construct a tailored database for each gene discovery project. Gene refining and structuring tools enable to reliably detect splice variants and antisense transcripts, generating understanding of various potential phenotypic outcomes of a single gene. The capabilities of the LEADS platform of Compugen LTD for analyzing human genome have been confirmed and accepted by the scientific committee (Widespread Antisense Transcription, Yelin, et al. (2003) Nature Biotechnology 21, 379-85; Splicing of Alu Sequences, Lev-Maor, et al. (2003) Science 300 (5623), 1288-91), and have proven most efficient in plant genomics as well.
EST clustering and gene assembly - For clustering and assembly of arabidopsis and rice genes the genomic LEADS version was employed. This tool allows most accurate clustering of ESTs and mRNA sequences on genome, and predicts gene structure as well as alternative splicing events and anti-sense transcription.
For organisms with no available full genome sequence data, expressed LEADS as well as TIGR (Hypertext Transfer Protocol ://World Wide Web (dot) tigr (dot) org/) clustering software were applied. The results of the two clustering tools were compared and in cases where clusters predicted by the two tools were significantly different, both versions were presented and considered.
Gene annotation - Predicted genes and proteins were annotated as follows:
• Blast search (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov (dot) library (dot) vu (dot) edu (dot) au/BLAST/ ) against all plant UniProt (Hypertext Transfer Protocol ://World Wide Web (dot) expasy (dot) uniprot (dot) org/) sequences was performed.
• Frame-Finder (Hypertext Transfer Protocol ://World Wide Web (dot) ebi (dot) ac (dot) uk/~guy/estate/) calculations with default statistics was used to predict protein sequences for each transcript.
2017228711 15 Sep 2017 • The predicted proteins were analyzed by InterPro (Hypertext Transfer Protocol://World Wide Web (dot) ebi (dot) ac (dot) uk/interpro/).
• Blast against proteins from AraCyc and ENZYME databases was used to map the predicted transcripts to AraCyc pathways.
• Each transcript was compared using tblastx algorithm (Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov (dot) library (dot) vu (dot) edu (dot) au/BLAST/) against all other organism databases to validate the accuracy of the predicted protein sequence, and for efficient detection of orthologs.
Gene expression profiling - Few data sources were exploited for gene expression profiling, namely microarray data and digital expression profile (see below). According to gene expression profile, a correlation analysis was performed to identify genes which are co-regulated under different development stages and environmental conditions.
Publicly available microarray datasets were downloaded from TAIR and NCBI GEO sites, renormalized, and integrated into the database. Expression profiling was one of the most important resource data for identifying genes important for ABST. Moreover, when homolog genes from different crops were responsive to ABST, the genes were marked as highly predictive to improve ABST.
A digital expression profile summary was compiled for each cluster according to all keywords included in the sequence records comprising the cluster. Digital expression, also known as electronic Northern Blot, is a tool that displays virtual expression profile based on the EST sequences forming the gene cluster. The tool can provide the expression profile of a cluster in terms of plant anatomy (in what tissues/organs is the gene expressed), developmental stage (the developmental stages at which a gene can be found) and profile of treatment (provides the physiological conditions under which a gene is expressed such as drought, cold, pathogen infection, etc). Given a random distribution of ESTs in the different clusters, the digital expression provides a probability value that describes the probability of a cluster having a total of N ESTs to contain X ESTs from a certain collection of libraries. For the probability calculations are taken into consideration: a) the number of ESTs in the cluster, b) the number of ESTs of the implicated and related libraries, c) the overall number of ESTs available representing the species. Thereby clusters with low probability values are highly enriched with ESTs from the group of libraries of interest indicating a specialized expression.
2017228711 15 Sep 2017
The concepts of orthology and paralogy have recently been applied to functional characterizations and classifications on the scale of whole-genome comparisons. Orthologs and paralogs constitute two major types of homologs: The first evolved from a common ancestor by specialization, and the latter are related by duplication events. It is assumed that paralogs arising from ancient duplication events are likely to have diverged in function while true orthologs are more likely to retain identical function over evolutionary time.
To further investigate and identify the ABST putative ortholog genes from monocot species, two computational methods were integrated:
(i) Method for alignments of ortholog sequences - based on construction ortholog groups across multiple eukaryotic taxa, using modifications on the Markov cluster algorithm to group putative orthologs and paralogs. These putative orthologs were further organized under Phylogram - a branching diagram (tree) assumed to be an estimate of a phylogeny of the genes.
(ii) Method for generating genes expression profile “Digital Expression ” The present inventors have performed considerable work aimed at annotating sequences. Expression data was analyzed and the EST libraries were classified using a fixed vocabulary of custom terms such as experimental treatments. The annotations from all the ESTs clustered to a gene were analyzed statistically by comparing their frequency in the cluster versus their abundance in the database, allowing to construct a numeric and graphic expression profile of that gene, which is termed “digital expression”.
The rationale of using these two complementary methods is based on the assumption that true orthologs are likely to retain identical function over evolutionary time. These two methods (sequence and expression pattern) provide two different sets of indications on function similarities between two homologous genes, similarities in the sequence level - identical amino acids in the protein domains and similarity in expression profiles.
Overall, 110 genes were identified to have a major impact on ABST when overexpressed in plants. The identified ABST genes, their curated polynucleotide and polypeptide sequences, as well as their updated sequences according to Genebank database are summarized in Table 1, hereinbelow.
Table 1
Identified ABST Genes
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
1 ΜΑΒΙ MAB1.0.ricegbl54BM4211 11 T1 rice 201
2 MAB1.1 .rice gb 157.2|BM42 1111T1 rice 202 updated to production gbl57.2 updated to production gbl57.2
3 MAB2 MAB2.0.rice|gbl54|AU2255 47_T1 rice No predicted protein
4 MAB2.1.rice|gbl57.2|AU225 547_T1 rice updated to production gbl57.2
5 MAB3 MAB3.0.rice|gbl54|BE03999 5 T1 rice 203
6 MAB3.1.ricegbl57.2BE039 995_T1 rice 204 updated to production gbl57.2 updated to production gbl57.2
7 MAB4 MAB4.0.rice|gbl54|B181227 7 T1 rice 205
8 MAB4.7.rice|gbl57.2|B18122 77 CT1 rice curated
9 MAB5 MAB5.0.rice|gbl54|CB6241 06 T1 rice 206
10 MAB6 MAB6.0.arabidopsis|gbl54|Z 47404 T1 arabidopsi s 207
11 MAB7 MAB7.0.arabidopsis|6|AT5G 47560.1 arabidopsi s 208
12 MAB7.1.arabidopsis|gbl65|A T5G47560 T1 arabidopsi s 209 updated to production gbl65 updated to production gbl65
13 MAB8 MAB8.0.rice|gbl54|BU6729 31 T1 rice 210
14 MAB8.7.rice|gbl54|BU6729 31_T1 rice Bioinformatics &DNA Curated
15 MAB9 MAB9.0.arabidopsis gbl54 B E844934 T1 arabidopsi s 211
16 MAB10 MAB10.0.arabidopsisgbl54 Z27056 T1 arabidopsi s 212
17 MAB11 MAB 11.O.arabidopsis gb 154 Z34014 T1 arabidopsi s 213
18 MAB 11.1 .arabidopsis|gb 1651 AT5G52300 T1 arabidopsi s 214 updated to production gbl65 updated to production gbl65
19 MAB12 MAB12.0.arabidopsisgbl54 ATLT1L40 T1 arabidopsi s 215
20 MAB 12.1 .arabidopsis|gb 1651 AT5G52310 T1 arabidopsi s 216 updated to production gbl65 updated to production gbl65
21 MAB13 MAB13.0.arabidopsis|6|AT2 G38760.1 arabidopsi s 217
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
22 MAB 13.1 .arabidopsis|gb 1651 AT2G38760 T1 arabidopsi s 218 updated to production gbl65 updated to production gbl65
23 MAB 14 MAB14.0.rice|gbl54|AB042 259 T1 rice 219
24 MAB14.1.ricegbl57.2 AB04 2259_T1 rice 220 updated to production gbl57.2 updated to production gbl57.2
25 MAB15 MAB 15.0. sorghum gb 15 4 Al 724695 T1 sorghum 221
26 MAB16 MAB16.0.rice|gbl54|B17951 72 T1 rice 222
27 MAB 16.1.rice gb 157.2 B1795 172_T1 rice 223 updated to production gbl57.2 updated to production gbl57.2
28 MAB17 MAB17.0.soybeangbl54 BE 821839 T1 soybean 224
29 MAB18 MAB 18.0.barley |gb 154 |BF62 5971 T1 barley 225
226 protein Bioinforma tics & Protein Curated
30 MAB19 M AB 19.0. sorghum gb 15 4 A W563861 T1 sorghum 227
31 MAB 19.1. sorghum|gb 161 .xe no|AW563861_Tl sorghum 228 updated to production gbl61.xeno updated to production gbl61.xeno
32 MAB20 MAB20.0.arabidopsisgbl54 T04691 T1 arabidopsi s 229
33 MAB20.1.arabidopsis|gbl65| AT1G61890 T1 arabidopsi s 230 updated to production gbl65 updated to production gbl65
34 MAB21 MAB21.0.ricegbl54BE2300 53 T1 rice 231
35 MAB21.1.rice|gbl57.2|BE23 0053_Tl rice 232 updated to production gbl57.2 updated to production gbl57.2
36 MAB22 MAB22.0.tomato|gbl54|BG7 91299 T1 tomato 233
234 Curated
37 MAB23 MAB23.0.rice|gbl54|B13058 10 T1 rice 235
38 MAB24 MAB24.0.rice|gbl54|B18082 73 T1 rice 236
39 MAB24.7.rice|gbl57.2|B1808 273 CT1 rice curated
40 MAB25 MAB25.0.arabidopsis|6|ATl G27760.1 arabidopsi s 237
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
41 MAB25.1 .arabidopsis|gb 1651 AT1G2776O_T1 arabidopsi s 238 updated to production gbl65 updated to production gbl65
42 MAB26 MAB26.0.rice|gbl54|AW155 625 T1 rice 239
43 MAB26.7.rice|gbl57.2|B1305 400 CT1 rice curated
44 MAB27 MAB27.0.arabidopsisgbl54 AY045660 T1 arabidopsi s 240
45 MAB27.7.arabidopsis|gbl65| AT5G24120 CT1 arabidopsi s curated
46 MAB28 MAB28.0.rice|gbl54|B17951 08 T1 rice 241
47 MAB28.7.rice|gbl57.2|B1795 108 CT1 rice curated
48 MAB29 MAB29.0.arabidopsisgbl54 AU239137 T2 arabidopsi s 242
49 MAB29.1 .arabidopsis|gb 1651 AT2G25600 T1 arabidopsi s 243 updated to production gbl65 updated to production gbl65
50 MAB30 MAB30.0.arabidopsisgbl54 AY062542 T1 arabidopsi s 244
51 MAB30.7.arabidopsis|gbl65| AT1G70300 CT1 arabidopsi s Curated
52 MAB31 MAB31.0. soybean gb 154 B19 68709 T1 soybean 245
53 MAB31.7.soybean|gbl62|B19 68709 CT1 soybean 246 Curated curated
54 MAB32 MAB32.0.rice|gbl54|AF0395 32 T1 rice 247
55 MAB33 MAB33.0.maize|gbl54|A161 5215 T1 maize 248
56 MAB 3 3.1. maize | gb 1641A161 5215_T1 maize 249 updated to production gbl64
57 MAB34 MAB34.0.barley|gbl54|TG BF625450 T1 barley 250
58 MAB34.1.barley gb 157.2 BF 62545O_T1 barley 251 updated to production gbl57.2 updated to production gbl57.2
59 MAB35 MAB35.0.arabidopsisgbl54 AA651513 T1 arabidopsi s 252
60 MAB3 5.1 .arabidopsis|gb 1651 AT2G16890 T1 arabidopsi s 253 updated to production gbl65 updated to production gbl65
61 MAB36 MAB36.0.arabidopsis|gbl54| AU239340 T1 arabidopsi s 254
62 MAB3 6.1 .arabidopsis|gb 1651 AT4G2757O_T1 arabidopsi s 255 updated to production gbl65 updated to production gbl65
63 MAB37 MAB37.0.tomato|gbl54|BGl 25939 T1 tomato 256
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
64 MAB37.7.tomato|gbl64|BGl 25939 CT1 tomato curated
65 MAB38 MAB38.0.wheat|gbl54|BE49 2836 T1 wheat 257
66 MAB38.7.wheat|gbl64|BE49 2836 CT1 wheat 258 curated curated
67 MAB39 MAB39.0.barley gbl54 AL5 00200 T1 barley 259
68 MAB39.1.barley gbl57.2 AL 500200_Tl barley 260 updated to production gbl57.2 updated to production gbl57.2
69 MAB40 MAB40.0.rice|gbl54|AA754 628 T1 rice 261
70 MAB40.7.rice|gbl57.2|AA75 4628 CT1 rice curated
71 MAB41 MAB41.0.tomato gb 154 A14 89494 T1 tomato 262
72 MAB41.7 .tomato | gb 1641A14 89494 CT1 tomato curated
73 MAB42 M AB 42.0. sorghum gb 15 4 Β E 595950 T1 sorghum 263
74 MAB42.7. sorghum|gb 161 .xe no|A1881418 CT1 sorghum 264 curated curated
75 MAB43 MAB43.0.arabidopsis gb 154 BE662945 T1 arabidopsi s 265
76 MAB43.1 .arabidopsis|gb 1651 AT5G26920 T1 arabidopsi s 266 updated to production gbl65 updated to production gbl65
77 MAB44 MAB44.0.arabidopsis gbl54 H36025 T1 arabidopsi s 267
78 MAB44.1 .arabidopsis|gb 1651 AT1G67360 T1 arabidopsi s 268 updated to production gbl65 updated to production gbl65
79 MAB45 MAB45.0.wheat|gbl54|TG BQ172359 T1 wheat 269
80 MAB45.1. wheat|gb 164|BQ 17 2359_T1 wheat 270 updated to production gbl64 updated to production gbl64
81 MAB46 MAB46.0.arabidopsis|gbl54| AA389812 T1 arabidopsi s 271
82 MAB47 M AB 4 7.0. sorghum gbl54 A W672286 T1 sorghum 272
83 M AB 4 7.7. sorghum| gb 161. xe no|A1948276 CT1 sorghum 273 Curated Curated
84 MAB48 MAB48.0.rice|gbl54|B18021 61 T1 rice 274
85 MAB48.7.rice|gbl57.2|AU09 2454 CT1 rice 275 curated curated
86 MAB49 MAB49.0.maize|gbl54|TG A1621810 T1 maize 276
87 MAB49.7.maize|gbl64|A162 1810 CTl maize Curated
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
88 MAB50 MAB50.0.arabidopsis gbl54 W43146 Tl arabidopsi s 277
89 MAB50.1 .arabidopsis|gb 1651 AT5G4857O_T1 arabidopsi s 278 updated to production gbl65 updated to production gbl65
90 MAB91 MAB91.0.arabidopsis gb 154 AU236480 Tl arabidopsi s 279
280 curated
91 MAB96 MAB96.0.arabidopsis|gbl54| Z27256 Tl arabidopsi s 281
92 MAB96.7.arabidopsis|gbl65| AT5G03800 CT1 arabidopsi s 282 curated curated
93 MAB99 MAB99.0.tomato|gbl54|BG7 35056 Tl tomato 283
94 MAB100 MAB100.0.arabidopsis gb 15 4|Z37259 Tl arabidopsi s 284
95 MAB 100.1 .arabidopsis|gb 16 5|AT1GO147O_T1 arabidopsi s 285 updated to production gbl65 updated to production gbl65
96 MAB104 MAB104.0.rice|gbl54|BE039 215 Tl rice 286
97 MAB 104.1 .rice gb 157.2|BE0 39215_T1 rice 287 updated to production gbl57.2 updated to production gbl57.2
98 MAB121 MAB 121.0. sugarcane gb 15 7 CA079500 Tl sugarcane 288
99 MAB 121.1. sugarcane gb 15 7. 2|CA079500_Tl sugarcane 289 updated to production gbl57.2 updated to production gbl57.2
100 MAB122 MAB122.0.maize|gbl54|A19 01344 T9 maize 290
101 MAB123 MAB123.0.barley|gbl57|BF6 26638 Tl barley 291
102 MAB123.1.barley|gbl57.2|B F626638_T1 barley 292 updated to production gbl57.2 updated to production gbl57.2
103 MAB124 MAB 124.0. sugarcane gb 157 CA284042 Tl sugarcane 293
104 MAB 124.1. sugarcane gb 15 7. 2|CA284042_Tl sugarcane 294 updated to production gbl57.2 updated to production gbl57.2
105 MAB125 MAB125.0.rice|gbl57|CF957 213 Tl rice 295
106 MAB 125.1 .rice gb 157.2|CF9 57213_T1 rice 296 updated to production gbl57.2 updated to production gbl57.2
107 MAB126 MAB126.0.grape|gbl57|BQ7 97309 Tl grape 297
108 MAB126.1.grape|gbl60|BQ7 97309_Tl grape 298 updated to production gbl60 updated to production gbl60
2017228711 15 Sep 2017
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
109 MAB127 MAB127.0.grape|gbl57|CB9 71532 Tl grape 299
110 MAB127.1.grape|gbl60|CB9 71532_T1 grape 300 updated to production gbl60 updated to production gbl60
111 MAB128 M AB 12 8.0. sugarcane gb 15 7 CA142162 Tl sugarcane 301
112 M AB 12 8.1. sugarcane gb 15 7. 2|CA142162_T1 sugarcane 302 updated to production gbl57.2 updated to production gbl57.2
113 MAB129 MAB129.0.tomato|gbl57|Al 486106 Tl tomato 303
114 MAB 129.1 .tomato | gb 1641 Al 4861O6_T1 tomato 304 updated to production gbl64 updated to production gbl64
115 MAB130 MAB130.0.canola|gbl57|CD 829694 Tl canola 305
116 MAB131 MAB 131.0.tomato gb 157 A W928843 Tl tomato 306
117 MAB131.1.tomato|gbl64|A W928843_T1 tomato 307 updated to production gbl64 updated to production gbl64
118 MAB132 MAB 132.0. barley gb 157 BF6 21624 Tl barley 308
119 MAB133 MAB133.0.barley|gbl57|BE4 11546 Tl barley 309
120 MAB133.1.barley|gbl57.2|B E411546T1 barley 310 updated to production gbl57.2 updated to production gbl57.2
121 MAB134 MAB 134.0. barley gb 157 BE4 37407 Tl barley 311
312 protein Bioinforma tics & Protein Curated
122 MAB135 MAB135.0.1otus|gbl57|A196 7693 Tl lotus 313
123 MAB135.1.1otusgbl57.2 A19 67693T1 lotus 314 updated to production gbl57.2 updated to production gbl57.2
124 MAB136 MAB136.0.rice|gbl57|AK05 8573 Tl rice 315
125 MAB136.1.ricegbl57.2 AKO 58573_T1 rice 316 updated to production gbl57.2 updated to production gbl57.2
126 MAB137 MAB137.0.barley|gbl57|AL 5O8624_T1 barley 317 from provisional patent
127 MAB137.1.barleygbl57.2 A L5O8624_T1 barley 318 updated to production gbl57.2 updated to production gbl57.2
2017228711 15 Sep 2017
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
128 MAB138 MAB138.0.potato|gbl57|Bll 77281_T1 potato 319 from provisional patent
129 MAB138.1.potato|gbl57.2|Bl 177281_T1 potato 320 updated to production gbl57.2 updated to production gbl57.2
130 MAB139 MAB139.0.cotton|gbl57.2|A 1727826_T1 cotton 321 from provisional patent
131 MAB 139.1 .cotton|gb 1641 Al 7 27826_T1 cotton 322 updated to production gbl64 updated to production gbl64
132 MAB140 MAB140.0.barley|gbl57|B17 78498_T1 barley 323 from provisional patent
133 MAB 140.1 .barley gb 157.2|B1 778498_T1 barley 324 updated to production gbl57.2 updated to production gbl57.2
134 MAB141 MAB141.0.barleygbl57BE4 21008_Tl barley 325 from provisional patent
135 MAB142 MAB142.0.cotton|gbl57.2|A 1055631_T2 cotton 326 from provisional patent
136 MAB142.0.cotton|gbl57.2|A 105563 IT1 cotton 327 from provisional patent
137 MAB142.1.cotton|gbl64|AW 187O41_T1 cotton 328 updated to production gbl64 updated to production gbl64
138 MAB143 MAB143.0.tomato|gbl57|Al 487157_T1 tomato 329 from provisional patent
139 MAB143.1.tomato|gbl64|Al 487157_T1 tomato 330 updated to production gbl64 updated to production gbl64
140 MAB144 MAB144.0.grape|gbl57|CA8 14960T1 grape 331 from provisional patent
141 MAB 144.1.grape gb 160 CA8 14960T1 grape 332 updated to production gbl60 updated to production gbl60
142 MAB145 MAB145.0.barley|gbl57|BE4 13365_T1 barley 333 from provisional patent
143 MAB146 MAB146.0.tomato|gbl57|Al 773927_T1 tomato 334 from provisional patent
144 MAB 146.1 .tomato | gb 1641 Al 773927_T1 tomato 335 updated to production gbl64 updated to production gbl64
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
145 MAB147 MAB147.0.tobacco|gbl57|E B446189 TI tobacco 336
146 MAB147.1.tobacco|gbl62|E B446189T1 tobacco 337 updated to production gbl62 updated to production gbl62
147 MAB148 M AB 14 8.0. medicago gb 15 7 AW256654 TI medicago 338
148 MAB 148.1.medicago gb 157. 2|AW256654_T1 medicago 339 updated to production gbl57.2 updated to production gbl57.2
149 MAB150 MAB150.0.canola|gbl57|CD 818831 TI canola 340
150 MAB 150.1 .canola|gb 161 |CD 818831T1 canola 341 updated to production gbl61 updated to production gbl61
151 MAB151 MAB151.0.potato|gbl57|BQ 513540 TI potato 342
152 MAB151.1.potato|gbl57.2|B Q513540T1 potato 343 updated to production gbl57.2 updated to production gbl57.2
153 MAB152 MAB152.0.grape|gbl57|BQ7 98655 TI grape 344
154 MAB152.1.grape|gbl60|BQ7 98655_T1 grape 345 updated to production gbl60 updated to production gbl60
155 MAB153 MAB 15 3.0. sugarcane gb 15 7 BQ533857 TI sugarcane 346
156 MAB 153.1 .sugarcane gb 157. 2|BQ533857_T1 sugarcane 347 updated to production gbl57.2 updated to production gbl57.2
157 MAB154 Μ AB 15 4.0. sugarc an e gb 15 7 BQ537570 T3 sugarcane 348
158 MAB 15 4.0. sugarc an e gb 15 7 BQ537570 T2 sugarcane 349
159 MAB 15 4.0. sugarc an e gb 15 7 BQ537570 TI sugarcane 350
160 MAB 15 4.1. sugarcane gb 15 7. 2|BQ537570_Tl sugarcane 351 updated to production gbl57.2 updated to production gbl57.2
161 MAB155 MAB 15 5.0. sorghum gb 15 7 A W676730 TI sorghum 352
162 MAB 155.1. sorghum|gb 161.x eno|AW676730_Tl sorghum 353 updated to production gbl61.xeno updated to production gbl61.xeno
163 MAB156 MAB156.0.tobacco|gbl57|A B117525 TI tobacco 354
164 MAB 156.1 .tobacco | gb 1621A B117525_T1 tobacco 355 updated to production gbl62 updated to production gbl62
165 MAB157 MAB 15 7.0. sugarcane gb 15 7 BQ533820 T2 sugarcane 356
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
166 MAB 15 7.0. sugarcane gb 15 7 BQ533820 T1 sugarcane 357
167 MAB 157.1. sugarcane gb 157. 2|BQ533820_Tl sugarcane 358 updated to production gbl57.2 updated to production gbl57.2
168 MAB 158 MAB158.0.cotton|gbl57.2|A 1054450 T1 cotton 359
169 MAB 159 MAB159.0.canola|gbl57|CD 818468 T1 canola 360
170 MAB 160 MAB 160.0.barley|gbl57|BF6 22450 T1 barley 361
171 MAB161 MAB 161.0.poplar|gb 157 |B U 896597 T1 poplar 362
172 MAB 161.1 .poplar|gb 15 7.2 |B U896597_T1 poplar 363 updated to production gbl57.2 updated to production gbl57.2
173 MAB 162 MAB 162.0. sugarcane | gb 15 71 BU102611 T1 sugarcane 364
174 MAB 162.1. sugarcane | gb 15 7. 2|BU 102611_T1 sugarcane 365 updated to production gbl57.2 updated to production gbl57.2
175 MAB 163 MAB163.0.barley|gbl57|AL 501813 T1 barley 366
176 MAB163.1.barley|gbl57.2|A L501813_Tl barley 367 updated to production gbl57.2 updated to production gbl57.2
177 MAB 164 MAB 164.0.barley|gbl57|BF2 53543 T1 barley 368
178 MAB164.1.barley|gbl57.2|B F253543_T1 barley 369 updated to production gbl57.2 updated to production gbl57.2
179 MAB 165 MAB165.0.grape|gbl57|BQ7 93123 T1 grape 370
180 MAB 166 MAB166.0.poplar|gbl57|CV 228694 T1 poplar 371
181 MAB166.1.poplar|gbl57.2|C V228694_T1 poplar 372 updated to production gbl57.2 updated to production gbl57.2
182 MAB 167 MAB167.0.canola|gbl57|CX 278043 T1 canola 373
183 MAB 167.1 .canola|gb 161 |CX 278O43_T1 canola 374 updated to production gbl61 updated to production gbl61
184 MAB 168 MAB168.0.grape|gbl57|BG2 73815 T1 grape 375
185 MAB168.1.grape|gbl60|BG2 73815_T1 grape 376 updated to production gbl60 updated to production gbl60
186 MAB 169 MAB169.0.cotton|gbl57.2|C OTLEA14BT1 cotton 377
2017228711 15 Sep 2017
SEQ ID NO: Polyn ucleot ide Gene Name Cluster Name Organism SEQ ID NO: Poly pepti de Polynucleotide Description Polypeptide Description
187 MAB169.1.cotton|gbl64|CO TLEA14BT1 cotton 378 updated to production gbl64 updated to production gbl64
188 ΜΑΒΙ 70 MAB170.0.barley|gbl57|BE4 12505 T1 barley 379
189 MAB 170.1.barley gb 157.2 B E4125O5_T1 barley 380 updated to production gbl57.2 updated to production gbl57.2
190 MAB171 M AB 171.0. sugarcane gb 15 7 CA123631 T1 sugarcane 381
191 MAB171.1 .sugarcane gb 157. 2|CA123631_T1 sugarcane 382 updated to production gbl57.2 updated to production gbl57.2
192 ΜΑΒΙ 72 MAB 172.0. sugarcane gb 15 7 BQ478980 T1 sugarcane 383
193 MAB 172.0. sugarcane gb 15 7 BQ478980 T2 sugarcane 384
194 MAB173 MAB 173.0. barley gb 157 BY 836652 T1 barley 385
195 MAB 173.1.barley gb 157.2 B Y836652_T1 barley 386 updated to production gbl57.2 updated to production gbl57.2
196 ΜΑΒΙ 74 MAB 174.0. barley gb 157 BG 342904 T1 barley 387
197 MAB 174.1.barley gb 157.2 B G342904T1 barley 388 updated to production gbl57.2 updated to production gbl57.2
198 MAB175 MAB175.0.tomato|gbl57|BG 126606 T1 tomato 389
199 MAB175.0.tomato|gbl57|BG 126606 T2 tomato 390
200 MAB 175.1.tomato|gbl64|BG 126606T1 tomato 391 updated to production gbl64 updated to production gbl64
1653 MAB66 MAB66.0.tomato|gbl64|BGl 24832 CT1 tomato 1651
Table 1.
Polynucleotides and polypeptides with significant homology to the identified ABST genes have been identified from the databases using BLAST software using the BlastX 5 algorithm. The query nucleotide sequences were SEQ ID NOs:l, 3, 5, 7, 9, 10, 11, 13,
15, 16, 17, 19, 21, 23, 25, 26, 28, 29, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 50, 52,
54, 55, 57, 59, 61, 63, 65, 67, 69, 71 ,73 ,75 ,77, 79, 81, 82, 84, 86, 88, 90, 91, 93, 94,
96, 98, 100, 101, 103, 105, 107, 109, 111, 113, 115, 116, 118, 119, 121, 122, 124, 126, 128, 130, 132, 134, 135, 138, 140, 142, 143, 145, 147, 149, 151, 153, 155, 157, 161, 10 163, 165, 168, 169, 170, 171, 173, 175, 177, 179, 180, 182, 184, 186, 188, 190, 192,
194, 196, 198 and 1653, and the identified ABST homologs are provided in Table 2, below.
2017228711 15 Sep 2017
Table 2 _________________________ABST Gene homologs
Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi
392 apple|gbl57.3|CN444532_Tl apple 961 Seq357.MAB157.15.sugarca ne 85
393 apple|gbl57.3|CN445371 T1 apple 962 Seq376.MAB 168.15.grape 87
394 apple|gbl57.3|CN878026_Tl apple 963 Seq350.MAB154.15.sugarca ne 80
395 apple|gbl57.3|CK900582 T1 apple 964 Seq321.MAB 139.15.cotton 85
396 apple gbl57.3CN888579 T2 apple 965 Seq256.MAB37.15.tomato 86
397 apple gbl57.3CN888579 T3 apple 966 Seq256.MAB37.15.tomato 81
398 apple gbl57.3CO066535 T1 apple 967 Seq370.ΜΑΒΙ 65.15.grape 84
399 apple gbl57.3CN888579 T1 apple 968 Seq256.MAB37.15.tomato 86
400 apple gb 157.3 CN496860 T1 apple 969 Seq321.ΜΑΒΙ 39.15.cotton 81
401 apricot|gbl57.2|BQ 134642 T1 apricot 970 Seq329.MAB 143.15.tomato 82
402 apricot|gbl57.2|CB822088 T1 apricot 971 Seq256.MAB37.15.tomato 88
403 aquilegia gb 157.3 DR915383 T1 aquilegia 972 Seq321.ΜΑΒΙ 39.15.cotton 83
404 aquilegia|gb 157.3 |DR913600 T1 aquilegia 973 Seq344.MAB 152.15.grape 83
405 aquilegia gb 157.3 DR920101 T1 aquilegia 974 Seq370.ΜΑΒΙ 65.15.grape 87
406 aquilegia gbl57.3 DT727583 T1 aquilegia 975 Seq311. MAB13 4.15 .barley 80
407 aquilegia gb 157.3 DR918523 T1 aquilegia 976 Seq376.ΜΑΒΙ 68.15.grape 82
408 arabidopsis|gbl65|ATlG6789 0 T2 arabidopsis 977 S eq263. MAB 42.15. sorghum 80
409 arabidopsis|gbl65|ATlG7807 0 T2 arabidopsis 978 S eq207. MAB 6.15. arabidop si s 97
410 arabidopsis|gb 1651 AT 1G5289 0 T3 arabidopsis 979 S eq211. MAB 9.15. arabidop si s 85
411 arabidopsis|gb 1651AT3G0662 0 T1 arabidopsis 980 Seq357.MAB157.15.sugarca ne 80
412 arabidopsis|gbl65|ATlG6789 0 T1 arabidopsis 981 S eq263. MAB 42.15. sorghum 80
413 arabidopsis|gbl65|AT5G1486 0 T1 arabidopsis 982 Seq341.MAB 150.15.canola 80
414 arabidopsis|gb 1651 AT 5G4947 0 T2 arabidopsis 983 S eq263. MAB 42.15. sorghum 81
415 arabidopsis|gb 1651 AT 5G4947 0 T1 arabidopsis 984 S eq263. MAB 42.15. sorghum 81
416 arabidopsis|gb 1651AT3G2417 0 T1 arabidopsis 985 Seq376.MAB 168.15.grape 80
417 arabidopsis|gbl65|ATlGl 167 0 Tl arabidopsis 986 Seq229.MAB20.15.arabidop sis 84
2017228711 15 Sep 2017
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
418 arabidopsis|gb 1651AT3G2523 0 T1 arabidopsis 987 Seq370.ΜΑΒΙ 65.15.grape 80
419 arabidopsis|gbl65|AT4G3250 0 T2 arabidopsis 988 Seq242.MAB29.15.arabidop sis 81
420 arabidopsis|gb 1651 AT 5G0676 0 T1 arabidopsis 989 Seq373.MAB 167.15.canola 84
421 arabidopsis|gb 1651 AT 4G2741 0 T3 arabidopsis 990 S eq211. MAB 9.15. arabidop si s 94
422 arabidopsis|gbl65|AT4G2756 0 T1 arabidopsis 991 Seq254.MAB36.15.arabidop sis 94
423 artemisia|gbl64|EY047508 T 1 artemisia 992 Seq321.MAB 139.15.cotton 80
424 artemisia|gbl64|EY060376 T 1 artemisia 993 Seq376.MAB 168.15.grape 85
425 artemisiagbl64 EY089381 T 1 artemisia 994 Seq256.MAB37.15.tomato 86
426 artemisia|gbl64|EY042537 T 1 artemisia 995 Seq349.MAB154.15.sugarca ne 80
427 b juncea|gb 164 |E VGN001020 08310737 T1 bjuncea 996 Seq360.MAB 159.15.canola 97
428 bjuncea gbl 64 EVGN084860 04170336 T1 bjuncea 997 Seq373.MAB 167.15.canola 94
429 bjuncea|gbl64|EVGN004299 14360666 T1 bjuncea 998 Seq370.MAB 165.15.grape 83
430 bjuncea gbl 64 EVGN002584 30752139P1 T1 bjuncea 999 Seq376.MAB 168.15.grape 80
431 bjuncea|gbl 64 |EVGN015689 09822952 T1 bjuncea 1000 Seq373.MAB 167.15.canola 98
432 b oleracea|gbl61|DY029719 T1 b oleracea 1001 Seq370.MAB 165.15.grape 82
433 b oleracea|gbl61|AM385106 T1 b oleracea 1002 Seq360.MAB 159.15.canola 96
434 b oleracea gbl61AM387179 T1 b oleracea 1003 Seq360.MAB 159.15.canola 91
435 b oleracea|gbl61|AM061306 T1 b oleracea 1004 Seq284.MAB100.15.arabido psis 86
436 b oleracea|gbl61|AB125639 T1 b oleracea 1005 Seq376.MAB 168.15.grape 80
437 b rapa|gbl62|EE523634 T1 b rap a 1006 Seq229.MAB20.15.arabidop sis 92
438 b_rapa|gb 162|EX024909_T 1 b rap a 1007 Seq217 .MAB 13.15. arabidop sis 83
439 b rapa|gbl62|EX070158 T2 b rap a 1008 S eq211. MAB 9.15. arabidop si s 95
440 b rapa|gbl62|CA992067 T1 b rap a 1009 Seq360.MAB 159.15.canola 94
441 b rapa|gbl62|EE520623 T1 b rap a 1010 Seq280.MAB91.10.arabidop sis 89
442 b_rapa|gb 1621C V545 896_T 1 b rap a 1011 S eq208. MAB 7.15. arabidop si s 88
443 b rapa|gbl62|CO749564 T1 b rap a 1012 Seq370.MAB 165.15.grape 82
444 b_rapa|gb 1621C V434105_T 1 b rap a 1013 Seq217 .MAB 13.15. arabidop sis 83
2017228711 15 Sep 2017
Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
445 b rapa|gbl62|AF008441 Tl b rap a 1014 Seq376.ΜΑΒΙ 68.15.grape 80
446 b rapa|gbl62|EX070158 Tl b rap a 1015 S eq211. MAB 9.15. arabidop si s 86
447 b rapa|gbl62|EX088727 Tl b rap a 1016 Seq271.MAB46.15.arabidop sis 93
448 b rapa|gbl62|BG544469 Tl b rap a 1017 Seq360.MAB 159.15.canola 82
449 b rapa|gbl62|DN962625 Tl b rap a 1018 Seq237.MAB25.15.arabidop sis 85
450 b_rapa|gb 1621C V544672_T 1 b rap a 1019 Seq284.MAB100.15.arabido psis 88
451 barley|gbl57.2|B1947678 Tl barley 1020 Seq3 6 8. MAB 164.15 .barley 92
452 barley|gbl57.2|AV835424 Tl barley 1021 Seq257.MAB38.15.wheat 97
453 barley|gbl57.2|BE455969 Tl barley 1022 Seq290.MAB 122.15.maize 84
454 barley gb 157.2 BE519575 T2 barley 1023 S eq263. MAB 42.15. sorghum 81
455 barley|gbl57.2|BF625959 Tl barley 1024 Seq221 .MAB 15.15. sorghum 83
456 barley|gbl57.2|BQ461470 Tl barley 1025 Seq356.MAB157.15.sugarca ne 82
457 basilicum|gbl57.3|DY333033 Tl basilicum 1026 Seq256.MAB37.15.tomato 87
458 bean|gbl64|CB542809 Tl bean 1027 Seq376.MAB 168.15.grape 80
459 bean|gbl64|CV529652 Tl bean 1028 Seq370.MAB 165.15.grape 83
460 bean|gbl64|CB543453 Tl bean 1029 Seq3 6 8. MAB 164.15 .barley 80
461 bean|gbl64|CV535253 Tl bean 1030 Seq256.MAB37.15.tomato 88
462 beet|gbl62|BQ592516 Tl beet 1031 Seq256.MAB37.15.tomato 86
463 beet|gbl62|BQ488223_Tl beet 1032 S eq211. MAB 9.15. arabidop si s 88
464 beet|gbl62|BQ583768 Tl beet 1033 Seq385.MAB173.15.barley 85
465 beet gb 162 BQ591963 Tl beet 1034 Seq3 6 8. MAB 164.15 .barley 80
466 brachypodium|gb 161 .xeno|BE 519575 Tl brachypodium 1035 Seq356.MAB157.15.sugarca ne 85
467 brachypodium|gb 161 .xeno|BG 368321 Tl brachypodium 1036 Seq247.MAB32.15.rice 81
468 brachypodium|gb 161 .xeno|BE 400652 Tl brachypodium 1037 Seq3 6 8. MAB 164.15 .barley 95
469 brachypodium|gb 161 .xeno|AL 502884 Tl brachypodium 1038 Seq210.MAB8.15.rice 82
470 brachypodium|gb 161 .xeno|B Y 836652 Tl brachypodium 1039 Seq385.MAB173.15.barley 90
471 brachypodium|gb 161 .xeno|BE 414917 Tl brachypodium 1040 Seq309.MAB 133.15.barley 93
472 brachypodium|gb 161 .xeno|BF 202085 Tl brachypodium 1041 Seq291 .MAB 123.15 .barley 83
473 brachypodium|gb 161 .xeno|BE 406378 Tl brachypodium 1042 Seq219.MAB14.15.rice 80
474 brachypodium|gb 161 .xeno|BE 517562 Tl brachypodium 1043 Seq366.MAB 163.15.barley 85
475 brachypodium|gb 161 .xeno|BE 420294 Tl brachypodium 1044 Seq290.MAB 122.15.maize 85
476 brachypodium|gb 161 .xeno|BG 369416 Tl brachypodium 1045 Seq270.MAB45.15. wheat 89
477 brachypodium|gb 161 .xeno|BE 406039 T2 brachypodium 1046 Seq241.MAB28.15.rice 93
2017228711 15 Sep 2017
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/yp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
478 brachypodium|gb 161 .xeno|BE 418087 TI brachypodium 1047 Seq325.MAB141.15.barley 86
479 brachypodium|gb 161 .xeno|BE 470780 TI brachypodium 1048 Seq221 .MAB 15.15. sorghum 81
480 brachypodium|gb 161 .xeno|AV 835424 TI brachypodium 1049 Seq257.MAB38.15.wheat 93
481 brachypodium|gb 161 .xeno|BE 398656 TI brachypodium 1050 Seq308.MAB 132.15.barley 93
482 brachypodium|gb 161 .xeno|BE 437407 TI brachypodium 1051 Seq311. MAB 13 4.15 .barley 98
483 brachypodium|gb 161 .xeno|BE 406039 T3 brachypodium 1052 Seq3 3 3 .MAB 145.15 .barley 81
484 brachypodium|gb 161 .xeno|BE 490408 TI brachypodium 1053 Seq264.MAB42.10.sorghum 80
485 brachypodium|gb 161 .xeno|BE 403745 TI brachypodium 1054 Seq379.MAB170.15.barley 92
486 brachypodium|gb 161 .xeno|BE 490591 TI brachypodium 1055 Seq366.MAB 163.15.barley 87
487 brachypodium|gb 161 .xeno|BQ 461470 T2 brachypodium 1056 Seq356.MAB157.15.sugarca ne 85
488 brachypodium|gb 161 .xeno|BE 517562 T2 brachypodium 1057 Seq366.MAB 163.15.barley 83
489 brachypodium|gb 161 .xeno|BE 413341 TI brachypodium 1058 Seq336.MAB 147.15.tobacco 80
490 brachypodium|gb 161 .xeno|BE 515529 TI brachypodium 1059 Seq259.MAB39.15.barley 96
491 brachypodium|gb 161 .xeno|D V 471778 TI brachypodium 1060 Seq348.MAB154.15.sugarca ne 83
492 canola|gbl61|EL587045 TI canola 1061 Seq277.MAB50.15.arabidop sis 87
493 canola|gb 161 |CX279297_T 1 canola 1062 Seq280.MAB91. lO.arabidop sis 85
494 canola|gbl61|CD815143 TI canola 1063 Seq222.MAB16.15.rice 80
495 canola|gbl61|CD831036_Tl canola 1064 Seq284.MAB100.15.arabido psis 86
496 canola|gbl61|EE466962 TI canola 1065 Seq360.MAB 159.15.canola 83
497 canola|gbl61|CN726580 TI canola 1066 Seq305.MAB 130.15.canola 89
498 canolagbl61 CD829644 TI canola 1067 Seq373.MAB 167.15.canola 86
499 canola|gb 1611A Y245 8 87_T 1 canola 1068 S eq211. MAB 9.15. arabidop si s 87
500 canola|gbl61|EE411591 TI canola 1069 S eq207. MAB 6.15. arabidop si s 88
501 canola|gbl61|DY020345 TI canola 1070 S eq211. MAB 9.15. arabidop si s 92
502 canola|gbl61|CD820718 TI canola 1071 Seq360.MAB 159.15.canola 95
503 canolagbl61 CX189134 TI canola 1072 Seq221 .MAB 15.15. sorghum 81
504 canola|gbl61|EG021120 TI canola 1073 Seq360.MAB 159.15.canola 83
505 canola|gb 161 |ES906182_T 1 canola 1074 Seq244.MAB 30.15 .arabidop sis 92
506 canola|gb 1611E S911977_T 1 canola 1075 Seq229.MAB20.15.arabidop sis 88
Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
507 canola|gb 161 |CD814410_T 1 canola 1076 Seq217 .MAB 13.15. arabidop sis 81
508 canola|gbl61|ES904177 T1 canola 1077 Seq208.MAB7.15.arabidopsi s 87
509 canola|gbl61|CD813775 T1 canola 1078 Seq370.MAB 165.15.grape 82
510 canola|gb 161 |CD824419_T 1 canola 1079 Seq229.MAB20.15.arabidop sis 94
511 canola|gbl61|CD825454 T1 canola 1080 Seq229.MAB20.15.arabidop sis 90
512 canola|gbl61|CD834184_Tl canola 1081 Seq284.MAB100.15.arabido psis 88
513 canola|gbl61|EE469078 T1 canola 1082 Seq370.MAB 165.15.grape 83
514 canola|gbl61|GFXAJ53511IX 1 T1 canola 1083 Seq305.MAB 130.15.canola 99
515 canola|gbl61|EE448267 T1 canola 1084 Seq222.MAB16.15.rice 80
516 canola|gb 161 |CX 193415_T 1 canola 1085 Seq237.MAB25.15.arabidop sis 85
517 canola|gbl61|CD813278 T1 canola 1086 Seq375.MAB 168.15.grape 80
518 castorbean gb 160 MDL28401 M000077 T1 castorbean 1087 Seq370.MAB 165.15.grape 86
519 castorbean gb 160 EE25 8294 T1 castorbean 1088 Seq256.MAB37.15.tomato 87
520 castorbean|gbl60|MDL28066 M000021 T1 castorbean 1089 Seq370.MAB 165.15.grape 85
521 castorbean|gbl60|AM267339 T1 castorbean 1090 Seq222.MAB16.15.rice 80
522 castorbean|gbl60|EG659656 T1 castorbean 1091 Seq376.MAB 168.15.grape 83
523 castorbean|gbl60|EG656754 T1 castorbean 1092 S eq263. MAB 42.15. sorghum 82
524 castorbean|gbl60|EE259826 T1 castorbean 1093 Seq3 62. MAB 161.15 .poplar 83
525 castorbean|gbl60|EG659299 T1 castorbean 1094 Seq300.MAB127.15.grape 81
526 castorbean|gbl60|EE259565 T1 castorbean 1095 Seq276.MAB49.15.maize 80
527 castorbean|gb 160|EE25513 3 T1 castorbean 1096 Seq321.MAB 139.15.cotton 84
528 castorbean|gbl60|MDL29822 M003364 T1 castorbean 1097 Seq336.MAB 147.15.tobacco 82
529 castorbean|gb 160|EG661241 T1 castorbean 1098 Seq371.MAB 166.15.poplar 85
530 centaurea|gbl61|EH713943 T 1 centaurea 1099 Seq321.MAB 139.15.cotton 82
531 centaurea gbl61EH724589 T 1 centaurea 1100 Seq256.MAB37.15.tomato 84
532 centaurea|gbl61|EH717520 T 1 centaurea 1101 Seq329.MAB 143.15.tomato 80
533 centaurea|gbl61|EH711566 T 1 centaurea 1102 Seq370.MAB 165.15.grape 81
534 centaurea|gbl61|EH713337 T 1 centaurea 1103 Seq259.MAB39.15.barley 81
Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
535 centaurea|gbl61|EH713628 T 1 centaurea 1104 Seq376.MAB 168.15.grape 83
536 centaurea|gbl61|EH738263 T 1 centaurea 1105 Seq385.MAB173.15.barley 80
537 centaurea|gbl61|EH727723 T 1 centaurea 1106 Seq256.MAB37.15.tomato 84
538 cichorium|gbl61|DT212291 T 1 cichorium 1107 Seq370.MAB 165.15.grape 80
539 cichorium gbl61DT211081 T 1 cichorium 1108 Seq376.MAB 168.15.grape 83
540 cichorium|gbl61|EH692437 T 1 cichorium 1109 Seq256.MAB37.15.tomato 86
541 cichorium|gbl61|DT212218 T 1 cichorium 1110 Seq256.MAB37.15.tomato 89
542 citrus|gbl57.2|CB290836 Tl citrus 1111 Seq376.MAB 168.15.grape 85
543 citrus|gbl57.2|BQ624861 Tl citrus 1112 Seq276.MAB49.15.maize 82
544 citrus gb 157.2 BQ624727 Tl citrus 1113 Seq370.MAB 165.15.grape 85
545 citrus|gbl57.2|CB290836 T2 citrus 1114 Seq376.MAB 168.15.grape 86
546 citrus|gbl57.2|CX672218_T2 citrus 1115 Seq357.MAB157.15.sugarca ne 83
547 citrus|gbl57.2|CF504250 Tl citrus 1116 Seq222.MAB16.15.rice 82
548 citrus|gbl57.2|CK933948 Tl citrus 1117 Seq256.MAB37.15.tomato 86
549 clover|gbl62|BB926896 Tl clover 1118 Seq256.MAB37.15.tomato 82
550 clover|gbl62|BB904696 Tl clover 1119 S eq263. MAB 42.15. sorghum 84
551 coffea|gbl57.2|DV676382 Tl coffea 1120 Seq256.MAB37.15.tomato 91
552 coffea gbl57.2 DV688680 Tl coffea 1121 Seq332.MAB 144.15.grape 83
553 coffea gbl57.2 DQ124044 Tl coffea 1122 Seq303.MAB 129.15.tomato 80
554 cotton|gbl64|BF268276 Tl cotton 1123 Seq370.MAB 165.15.grape 84
555 cotton gb 164 CO 113031 Tl cotton 1124 Seq319. MAB 138.15 .potato 80
556 cotton|gbl64|A1730186 Tl cotton 1125 Seq256.MAB37.15.tomato 81
557 cotton|gbl64|C0103100 Tl cotton 1126 Seq256.MAB37.15.tomato 86
558 cotton|gbl64|BE051970 Tl cotton 1127 Seq370.MAB 165.15.grape 84
559 cotton|gbl64|A1725698 Tl cotton 1128 Seq376.MAB 168.15.grape 85
560 cotton|gbl64|A1728290 Tl cotton 1129 Seq370.MAB 165.15.grape 82
561 cotton|gbl64|A1055482 Tl cotton 1130 Seq370.MAB 165.15.grape 85
562 cotton|gbl64|ES794517 Tl cotton 1131 Seq327.MAB 142.15.cotton 81
563 cotton|gbl64|BF268276 T2 cotton 1132 Seq370.MAB 165.15.grape 84
564 cotton|gbl64|CO109448 Tl cotton 1133 Seq376.MAB 168.15.grape 83
565 cotton|gbl64|DT459182 Tl cotton 1134 Seq375.MAB 168.15.grape 84
566 cotton|gbl64|BG441162 Tl cotton 1135 Seq256.MAB37.15.tomato 85
567 cowpea gbl65 FF390508 Tl cowpea 1136 Seq256.MAB37.15.tomato 84
568 cowpea|gbl65|FF390203 Tl cowpea 1137 Seq259.MAB39.15.barley 86
569 cowpea|gbl65|DQ267475 Tl cowpea 1138 Seq376.MAB 168.15.grape 83
570 cowpea gb 165 FF382851 Tl cowpea 1139 Seq224.MAB 17.15.soybean 89
571 cowpea|gbl65|FF394009 Tl cowpea 1140 Seq370. MAB 165.15. grape 85
572 dandelion|gbl61|DQ160099 T 1 dandelion 1141 Seq376.MAB 168.15.grape 82
573 dandeliongbl61 DY823013 T 1 dandelion 1142 Seq256.MAB37.15.tomato 82
574 dandeliongbl61 DY820394 T 2 dandelion 1143 Seq256.MAB37.15.tomato 88
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
575 dandeliongbl61 DY813450 T 2 dandelion 1144 Seq256.MAB37.15.tomato 85
576 dandeliongbl61 DY820394 T 1 dandelion 1145 Seq256.MAB37.15.tomato 87
577 fescue|gbl61|DT687914 TI fescue 1146 Seq290.MAB 122.15.maize 93
578 fescuegbl61 DT702477 TI fescue 1147 Seq291 .MAB 123.15 .barley 87
579 fescue gbl61 DT705881 TI fescue 1148 Seq311. MAB 13 4.15 .barley 96
580 fescue|gbl61|DT682501 TI fescue 1149 Seq321.MAB 139.15.cotton 82
581 fescue|gbl61|DT699000 TI fescue 1150 Seq309.MAB 133.15.barley 90
582 fescue|gbl61|DT706685 TI fescue 1151 Seq259.MAB39.15.barley 96
583 fescue|gbl61|DT698326 TI fescue 1152 Seq3 6 8. MAB 164.15 .barley 95
584 fescuegbl61 DT677453 TI fescue 1153 Seq379.MAB170.15.barley 95
585 fescuegbl61 DT674734 TI fescue 1154 Seq3 3 3 .MAB 145.15 .barley 88
586 ginger|gbl64|DY377113 TI ginger 1155 Seq223.MAB16.15.rice 81
587 grape gb 160 BQ792651 TI grape 1156 Seq222.MAB16.15.rice 84
588 grape gb 160 BQ793581 TI grape 1157 Seq371.MAB 166.15.poplar 80
589 iceplant|gbl64|BM658279 TI iceplant 1158 Seq376.MAB 168.15.grape 83
590 iceplant|gbl64|BE034140 TI iceplant 1159 Seq303.MAB 129.15.tomato 81
591 ipomoea|gbl57.2|AU224303 TI ipomoea 1160 Seq256.MAB37.15.tomato 91
592 ipomoea|gbl57.2|AU224807 TI ipomoea 1161 Seq385.MAB173.15.barley 80
593 ipomoea|gbl57.2|CJ758382 T 1 ipomoea 1162 Seq371.MAB 166.15.poplar 83
594 lettuce|gbl57.2|DW048067 T 1 lettuce 1163 Seq256.MAB37.15.tomato 87
595 lettuce|gbl57.2|DW046482 T 1 lettuce 1164 Seq256.MAB37.15.tomato 85
596 lettuce|gbl57.2|DW062524 T 1 lettuce 1165 Seq259.MAB39.15.barley 81
597 lettuce|gbl57.2|DW048641 T 1 lettuce 1166 Seq370.MAB 165.15.grape 80
598 lettuce|gbl57.2|DW055618 T 1 lettuce 1167 Seq3 71. MAB 166.15 .poplar 80
599 lettuce gb 157.2 DY961700_T2 lettuce 1168 S eq211. MAB 9.15. arabidop si s 83
600 lettuce gb 157.2 DW075962 T 1 lettuce 1169 Seq256.MAB37.15.tomato 87
601 lettuce|gbl57.2|DW047202 T 1 lettuce 1170 Seq376.MAB 168.15.grape 83
602 lotus|gbl57.2|BF177835 TI lotus 1171 Seq256.MAB37.15.tomato 90
603 lotus|gbl57.2|BW601503_Tl lotus 1172 S eq211. MAB 9.15. arabidop si s 84
604 maize|gbl64|T15319 T2 maize 1173 Seq276.MAB49.15.maize 96
605 maize|gbl64|A1649734 TI maize 1174 Seq264.MAB42.10.sorghum 90
606 maize|gbl64|BE638692 TI maize 1175 S eq228. MAB 19.15. sorghum 88
607 maize|gbl64|AW498283 TI maize 1176 Seq210.MAB8.15.rice 80
608 maize|gbl64|A1622375 TI maize 1177 Seq309.MAB 133.15.barley 90
609 maize|gbl64|BQ034409 TI maize 1178 Seq290.MAB 122.15.maize 100
610 maize|gbl64|EC895235 TI maize 1179 Seq210.MAB8.15.rice 86
611 maize|gbl64|A1947795 T2 maize 1180 Seq325.MAB141.15.barley 80
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
612 maize|gbl64|A1947974 T1 maize 1181 Seq227.MAB 19.15.sorghum 93
613 maize|gbl64|A1619086 T1 maize 1182 S eq3 46. M AB 15 3.15. sugarca ne 95
614 maize|gbl64|AA143925 T1 maize 1183 Seq221 .MAB15.15. sorghum 94
615 maize|gbl64|AW179463 T1 maize 1184 Seq321.ΜΑΒΙ 39.15.cotton 82
616 maize|gbl64|BE051802 T1 maize 1185 Seq231.MAB21.15.rice 89
617 maize|gbl64|A1942091 T1 maize 1186 Seq309.MAB 133.15.barley 89
618 maize|gbl64|A1944064 T1 maize 1187 Seq383.MAB 172.15. sugarca ne 96
619 maize|gbl64|T15319 T1 maize 1188 Seq276.MAB49.15.maize 96
620 maize|gbl64|A1782993 T1 maize 1189 Seq241.MAB28.15.rice 82
621 maize|gbl64|T26945 T1 maize 1190 Seq370.MAB 165.15.grape 80
622 maize|gbl64|A1941749 T1 maize 1191 Seq269.MAB45.15. wheat 91
623 maize|gbl64|A1891255 T1 maize 1192 Seq311. MAB 13 4.15 .barley 95
624 maize|gbl64|CD975046 T1 maize 1193 Seq203.MAB3.15.rice 88
625 maize|gbl64|AW360563 T1 maize 1194 Seq241.MAB28.15.rice 81
626 maize|gbl64|A1901860 T1 maize 1195 Seq259.MAB39.15.barley 85
627 maize gbl64 A1948098_Tl maize 1196 Seq3 81 .MAB 171.15. sugarca ne 95
628 maize|gbl64|A1444730 T1 maize 1197 Seq241.MAB28.15.rice 83
629 maize|gbl64|AW216308 T1 maize 1198 Seq288.MAB 121.15.sugarca ne 89
630 maize|gbl64|BM268089 T1 maize 1199 Seq3 81 .MAB 171.15. sugarca ne 92
631 maize|gbl64|A1438597 T1 maize 1200 S eq3 52. MAB 15 5.15. sorghu m 91
632 maize|gbl64|AW927739 T1 maize 1201 Seq350.MAB 154.15.sugarca ne 97
633 maize|gbl64|A1891255 T2 maize 1202 Seq311. MAB 13 4.15 .barley 95
634 maize|gbl64|A1920760 T1 maize 1203 Seq286.MAB104.15.rice 89
635 medicago gbl57.2 A1974487 T1 medicago 1204 Seq370.MAB 165.15.grape 87
636 medicago|gbl57.2|BE325770 T1 medicago 1205 Seq256.MAB37.15.tomato 88
637 medicago|gbl57.2|AW685603 T1 medicago 1206 Seq376.MAB 168.15.grape 82
638 medicago|gbl57.2|AL368329 T1 medicago 1207 Seq311. MAB 13 4.15 .barley 80
639 medicago gbl 57.2 AW688497 T1 medicago 1208 Seq370.MAB 165.15.grape 80
640 medicago gbl57.2 AL377093 T1 medicago 1209 Seq224.MAB 17.15.soybean 80
641 medicago gbl57.2 A1974241 T1 medicago 1210 Seq334.MAB 146.15.tomato 83
642 medicago|gbl57.2|BF632135 T1 medicago 1211 Seq344.MAB 152.15.grape 85
643 melon|gbl65|DV633691 T1 melon 1212 Seq376.MAB 168.15.grape 80
644 melon|gbl65|DV632564 T1 melon 1213 Seq3 6 8. MAB 164.15 .barley 80
645 melon|gbl65|DV633584 T1 melon 1214 Seq344.MAB 152.15.grape 86
646 melon|gbl65|AM714958 Tl melon 1215 Seq259.MAB39.15.barley 81
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/yp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
647 nicotiana benthamiana|gbl62| EH364164 T1 nicotiana bent hamiana 1216 Seq256.MAB37.15.tomato 95
648 oat|gbl64|CN816769 T1 oat 1217 Seq3 6 8. MAB164.15 .barley 94
649 oat|gbl64|BE439108 T1 oat 1218 Seq312.MAB134.10.barley 85
650 onion|gbl62|CF437899 T1 onion 1219 Seq256.MAB37.15.tomato 81
651 onion gbl62 CF437716 T1 onion 1220 Seq276.MAB49.15.maize 82
652 onion gbl62 CF439314 T1 onion 1221 Seq370.MAB 165.15.grape 80
653 papaya|gbl65|EX245596 T1 papaya 1222 Seq370.MAB 165.15.grape 88
654 papaya gb 165 EX299345 T1 papaya 1223 Seq263.MAB42.15.sorghum 82
655 papaya gb 165 EX248971 T1 papaya 1224 Seq3 62. MAB 161.15 .poplar 86
656 papaya|gbl65|EX227965 T1 papaya 1225 Seq332.MAB 144.15.grape 83
657 papaya|gbl65|EX264060 T1 papaya 1226 Seq376.MAB 168.15.grape 89
658 papaya|gbl65|EX291966 T1 papaya 1227 Seq370.MAB 165.15.grape 82
659 peach|gbl57.2|BU039922 T1 peach 1228 Seq300.MAB127.15.grape 82
660 peachgbl57.2BU039373 T1 peach 1229 Seq370.MAB 165.15.grape 83
661 peach|gbl57.2|AJ631618 T1 peach 1230 Seq276.MAB49.15.maize 80
662 peach|gbl57.2|BU040470 T1 peach 1231 Seq376.MAB 168.15.grape 89
663 peach gbl57.2 BU039381 T1 peach 1232 Seq256.MAB37.15.tomato 88
664 peanut|gbl61|ES754023 T1 peanut 1233 Seq332.MAB 144.15.grape 80
665 peanut|gbl61|EH043199 T1 peanut 1234 Seq256.MAB37.15.tomato 88
666 pepper gbl57.2 BM063531 T 1 pepper 1235 Seq256.MAB37.15.tomato 96
667 pepper|gbl57.2|BM062846 T 1 pepper 1236 Seq221 .MAB 15.15. sorghum 82
668 peppergbl57.2BM061776 T 1 pepper 1237 Seq329.MAB 143.15.tomato 90
669 pepper|gbl57.2|BM064151 T 1 pepper 1238 Seq306.MAB131.15.tomato 88
670 peppergbl57.2BM061313 T 1 pepper 1239 S eq211. MAB 9.15. arabidop si s 86
671 pepper gb 157.2 B1480604 T1 pepper 1240 Seq276.MAB49.15.maize 80
672 peri winkle | gb 164|EG559012 T1 periwinkle 1241 Seq259.MAB39.15.barley 80
673 petunia gbl57.2 CV292753 T 1 petunia 1242 S eq263. MAB 42.15. sorghum 80
674 petunia|gbl57.2|CV298220 T 1 petunia 1243 Seq283.MAB99.15.tomato 81
675 pine|gbl57.2|DR088714_Tl pine 1244 Seq357.MAB157.15.sugarca ne 80
676 pine|gbl57.2|AW290504 T1 pine 1245 Seq344.MAB 152.15.grape 82
677 pineapple gb 157.2 CO731309 T1 pineapple 1246 Seq222.MAB16.15.rice 83
678 pineapple|gbl57.2|DT336648 T1 pineapple 1247 Seq376.MAB 168.15.grape 81
679 pineapple gb 157.2 CO731994 T1 pineapple 1248 Seq219.MAB14.15.rice 80
680 poplar|gbl57.2|A1162293 T1 poplar 1249 Seq298.MAB 126.15.grape 82
681 poplar|gbl57.2|A1165439 T1 poplar 1250 Seq298.MAB 126.15.grape 80
682 poplar|gbl57.2|A1162293 T3 poplar 1251 Seq298.MAB 126.15.grape 80
683 poplar|gbl57.2|B1120274 T3 poplar 1252 Seq256.MAB37.15.tomato 81
684 poplarjgb 157.2 B1120274 T2 poplar 1253 Seq344.MAB 152.15.grape 89
Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
685 poplar|gbl57.2|BF299457 T1 poplar 1254 Seq370.ΜΑΒΙ 65.15.grape 85
686 poplar|gbl57.2|B1120274 T1 poplar 1255 Seq344.MAB 152.15.grape 86
687 poplar|gbl57.2|B1122516 T1 poplar 1256 Seq3 62. MAB161.15 .poplar 90
688 poplar|gbl57.2|BU821689 T1 poplar 1257 Seq321. ΜΑΒΙ 39.15. cotton 81
689 poplar|gbl57.2|A1166955 T1 poplar 1258 Seq344.MAB 152.15.grape 87
690 poplar|gbl57.2|B1069450 T1 poplar 1259 Seq376.MAB 168.15.grape 85
691 potato|gbl57.2|BG594910 T1 potato 1260 Seq370.MAB 165.15.grape 82
692 potato|gbl57.2|AJ487418 T1 potato 1261 Seq321. MAB 139.15. cotton 82
693 potato|gbl57.2|BQ516076 T2 potato 1262 Seq3 89.MAB 175.15.tomato 97
694 potato|gbl57.2|BE921143_Tl potato 1263 S eq3 49. MAB 15 4.15. sugarca ne 80
695 potato|gbl57.2|BG592541 T1 potato 1264 Seq256.MAB37.15.tomato 90
696 potato|gbl57.2|BF052848 T1 potato 1265 Seq321. MAB 139.15. cotton 81
697 potato gb 157.2 BF460150 T1 potato 1266 Seq370.MAB 165.15.grape 84
698 potato|gbl57.2|BG097985 T1 potato 1267 Seq303.MAB 129.15.tomato 91
699 potato|gbl57.2|BE923564 T1 potato 1268 Seq342. MAB 151.15 .potato 90
700 potato|gbl57.2|X86021 T1 potato 1269 Seq334.MAB 146.15.tomato 97
701 potato|gbl57.2|BG594768 T1 potato 1270 Seq329.MAB 143.15.tomato 97
702 potato|gbl57.2|BF154203 T1 potato 1271 Seq256.MAB37.15.tomato 98
703 potato|gbl57.2|BE344306 T1 potato 1272 Seq357.MAB 157.15. sugarca ne 82
704 potato|gbl57.2|BF460309 T1 potato 1273 Seq329.MAB 143.15.tomato 98
705 potato|gbl57.2|BQ516076 T1 potato 1274 Seq3 90.MAB 175.15 .tomato 96
706 potato|gbl57.2|B1176616 T1 potato 1275 Seq256.MAB37.15.tomato 88
707 potato|gbl57.2|BQ 117692 T1 potato 1276 Seq354.MAB 156.15.tobacco 86
708 potato|gbl57.2|AJ487418 T2 potato 1277 Seq321. MAB 139.15. cotton 81
709 potato|gbl57.2|BG351229_Tl potato 1278 Seq357.MAB 157.15. sugarca ne 81
710 potato|gbl57.2|AJ487418 T3 potato 1279 Seq321. MAB 139.15. cotton 84
711 potato|gbl57.2|BF154154 T1 potato 1280 Seq256.MAB37.15.tomato 99
712 radish|gbl64|EY895633 T1 radish 1281 Seq373. MAB 167.15. canola 93
713 radish|gb 164|EX772944_T 1 radish 1282 Seq356.MAB 157.15. sugarca ne 83
714 radish|gbl64|EW725846 T1 radish 1283 Seq237.MAB25.15.arabidop sis 84
715 radish|gbl64|EV527306 T1 radish 1284 Seq229.MAB20.15.arabidop sis 94
716 radish|gbl64|EV565850 T1 radish 1285 Seq277.MAB50.15.arabidop sis 90
717 radish|gbl64|EX772722 T1 radish 1286 Seq360. MAB 159.15. canola 88
718 radish gb 164 EX775718 T1 radish 1287 Seq376.MAB 168.15.grape 81
719 radishgbl64 EV535278 T1 radish 1288 Seq360. MAB 159.15. canola 81
720 radish|gbl64|EV565334 T1 radish 1289 S eq211. MAB 9.15. arabidop si s 91
721 radish gbl 64 EV528083_Tl radish 1290 Seq252.MAB35.15.arabidop sis 80
722 radish|gbl64|T25168 T1 radish 1291 Seq376.MAB 168.15.grape 80
723 radish|gbl64|EV544010 T1 radish 1292 Seq229.MAB20.15 .arabidop sis 91
724 radish|gb 164 |E W713 752 T 1 radish 1293 Seq373. MAB 167.15. canola 86
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
725 radish|gbl64|EV568565 T1 radish 1294 Seq284.MAB100.15.arabido psis 88
726 radish|gbl64|EV543867 T1 radish 1295 Seq373. MAB 167.15. canola 88
727 radish|gb 164|EX770974_T 1 radish 1296 S eq211. MAB 9.15. arabidop si s 85
728 radish|gbl64|EV566819 T1 radish 1297 Seq217 .MAB 13.15. arabidop sis 81
729 rice|gbl57.2|NM001059403 T 1 rice 1298 Seq261.MAB40.15.rice 84
730 rice|gbl57.2|C28755 T1 rice 1299 Seq321.MAB 139.15.cotton 80
731 rice|gbl57.2|AA750806 T1 rice 1300 Seq290.MAB 122.15.maize 83
732 rice gb 157.2 AA751345 T1 rice 1301 Seq321.MAB 139.15.cotton 80
733 rice|gbl57.2|BE040195_T6 rice 1302 Seq346.MAB153.15.sugarca ne 95
734 rice|gbl57.2|Bll 18752 T1 rice 1303 Seq276.MAB49.15.maize 94
735 rice|gbl57.2|AW070148_Tl rice 1304 Seq350.MAB154.15.sugarca ne 87
736 rice|gbl57.2|AW069929 T1 rice 1305 Seq309.MAB 133.15.barley 93
737 ricegbl57.2 AW070094 T1 rice 1306 Seq274.MAB48.15.rice 83
738 rice|gbl57.2|AA753115 T4 rice 1307 Seq259.MAB39.15.barley 90
739 rice|gbl57.2|B1795037 T4 rice 1308 Seq385.MAB173.15.barley 100
740 rice|gbl57.2|AU092454 T1 rice 1309 Seq274.MAB48.15.rice 100
741 rice gbl57.2 AA753115 T3 rice 1310 Seq259.MAB39.15.barley 91
742 rice|gbl57.2|BE040195_Tl rice 1311 Seq346.MAB153.15.sugarca ne 91
743 rice|gbl57.2|CB624284 T1 rice 1312 Seq264.MAB42.10.sorghum 82
744 rice|gbl57.2|AU030125_T3 rice 1313 Seq357.MAB157.15.sugarca ne 88
745 rice|gbl57.2|ALH64313 T1 rice 1314 Seq270.MAB45.15. wheat 84
746 rice|gbl57.2|B1799463 T1 rice 1315 Seq221 .MAB 15.15. sorghum 85
747 rice|gbl57.2|AW070094 T3 rice 1316 Seq274.MAB48.15.rice 80
748 rice|gbl57.2|AA753115 T1 rice 1317 Seq259.MAB39.15.barley 91
749 rice|gbl57.2|AU093322 T2 rice 1318 S eq228. MAB 19.15. sorghum 85
750 rice|gbl57.2|AU030125 T1 rice 1319 S eq263. MAB 42.15. sorghum 80
751 rice gb 157.2 AA752703 T1 rice 1320 Seq295.MAB125.15.rice 88
752 rice|gbl57.2|NM001067464 T 1 rice 1321 Seq205.MAB4.15.rice 93
753 rice|gbl57.2|NM001052309 T 1 rice 1322 Seq295.MAB125.15.rice 91
754 rice|gbl57.2|CA763128 T2 rice 1323 Seq219.MAB14.15.rice 80
755 rice|gbl57.2|AW070148_T2 rice 1324 Seq348.MAB154.15.sugarca ne 87
756 rice|gbl57.2|AU093322 T1 rice 1325 S eq228. MAB 19.15. sorghum 86
757 rice gbl57.2 AA753115 T5 rice 1326 Seq259.MAB39.15.barley 94
758 rice|gbl57.2|AU030125 T4 rice 1327 S eq263. MAB 42.15. sorghum 80
759 rye|gbl64|BF429408 T1 rye 1328 Seq309.MAB 133.15.barley 97
760 rye|gbl64|BE494847 T1 rye 1329 Seq3 6 8. MAB 164.15 .barley 97
761 safflower|gbl62|EL373402 T 1 safflower 1330 Seq376.MAB 168.15.grape 81
762 safflower|gbl62|EL374175 T 1 safflower 1331 Seq259.MAB39.15.barley 83
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/yp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
763 safflower|gbl62|EL377332 T 1 safflower 1332 Seq385.MAB173.15.barley 81
764 safflower gb 162 EL3 73487 T 1 safflower 1333 S eq263. MAB 42.15. sorghum 80
765 safflower|gbl62|EL374095 T 1 safflower 1334 Seq256.MAB37.15.tomato 86
766 safflower|gbl62|EL3 82051 T 1 safflower 1335 Seq256.MAB37.15.tomato 86
767 safflowergbl62EL409148 T 1 safflower 1336 Seq385.MAB173.15.barley 80
768 sorghum|gb 161. xeno | A W2249 27 Tl sorghum 1337 Seq288.MAB121.15.sugarca ne 94
769 sorghum|gb 161 .xeno |T26945 T2 sorghum 1338 Seq370.MAB 165.15.grape 81
770 sorghum|gb 161 .xeno|A193217 9 T3 sorghum 1339 Seq286.MAB104.15.rice 91
771 sorghum|gb 161. xeno |T 15 319 Tl sorghum 1340 Seq276.MAB49.15.maize 97
772 sorghum|gb 161 .xeno | Al 61521 5 Tl sorghum 1341 Seq248.MAB33.15.maize 92
773 sorghum|gb 161 .xeno|BG 1020 66 T2 sorghum 1342 Seq290.MAB 122.15.maize 90
774 sorghum| gb 161. xeno | AW6724 19 T2 sorghum 1343 Seq276.MAB49.15.maize 97
775 sorghum| gb 161. xeno | AW6724 19 T3 sorghum 1344 Seq276.MAB49.15.maize 95
776 sorghum|gb 161. xeno | A19018 6 0 Tl sorghum 1345 Seq259.MAB39.15.barley 84
777 sorghum|gb 161 .xeno | A162199 5 T3 sorghum 1346 Seq384.MAB172.15.sugarca ne 97
778 sorghum gb 161 .xeno A18 8141 8 T2 sorghum 1347 Seq264.MAB42.10.sorghum 100
779 sorghum gb 161 .xeno Al 8 9125 5 Tl sorghum 1348 Seq311. MAB 13 4.15 .barley 95
780 sorghum gb 161 .xeno A178299 3 Tl sorghum 1349 Seq241.MAB28.15.rice 84
781 sorghum|gb 161 .xeno | A172462 9 Tl sorghum 1350 Seq350.MAB154.15.sugarca ne 99
782 sorghum|gb 161 .xeno| AA1439 25 Tl sorghum 1351 Seq221 .MAB 15.15. sorghum 100
783 sorghum|gb 161 .xeno | A162199 5 T2 sorghum 1352 Seq383.MAB172.15.sugarca ne 99
784 sorghum|gb 161 .xeno |T26945 Tl sorghum 1353 Seq370.MAB 165.15.grape 81
785 sorghum|gb 161. xeno | A W1794 63 Tl sorghum 1354 Seq321.MAB 139.15.cotton 80
786 sorghum|gb 161 .xeno|ZMU909 44 T2 sorghum 1355 Seq367.MAB 163.15.barley 80
787 sorghum|gb 161. xeno |T 15 319 T2 sorghum 1356 Seq276.MAB49.15.maize 95
788 sorghum|gb 161 .xeno | A162199 5 T1 sorghum 1357 Seq383.MAB172.15.sugarca ne 99
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/yp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
789 sorghum|gb 161 .xeno|A193217 9 TI sorghum 1358 Seq286.MAB104.15.rice 90
790 sorghum|gb 161 .xeno | A162199 5 T4 sorghum 1359 Seq383.MAB172.15.sugarca ne 99
791 sorghum|gb 161 .xeno|ZMU909 44 T3 sorghum 1360 Seq367.MAB 163.15.barley 80
792 sorghum|gb 161 .xeno| A166522 9 T2 sorghum 1361 Seq346.MAB153.15.sugarca ne 96
793 sorghum gb 161. xeno A193 9 8 3 6 TI sorghum 1362 Seq309.MAB 133.15.barley 92
794 sorghum|gb 161 .xeno|B109906 8 TI sorghum 1363 Seq270.MAB45.15. wheat 83
795 sorghum|gb 161 .xeno | A166522 9 TI sorghum 1364 Seq346.MAB153.15.sugarca ne 96
796 sorghum| gb 161. xeno | AW6724 19 TI sorghum 1365 Seq276.MAB49.15.maize 97
797 sorghum|gb 161. xeno | A W4982 83 TI sorghum 1366 Seq210.MAB8.15.rice 83
798 sorghum|gb 161. xeno | A W9237 75 TI sorghum 1367 Seq231.MAB21.15.rice 88
799 sorghum|gb 161. xeno |T 15 319 T3 sorghum 1368 Seq276.MAB49.15.maize 85
800 soybeangbl62BG839539 TI soybean 1369 Seq3 6 8. MAB 164.15 .barley 80
801 soybeangb 162 CA783290 TI soybean 1370 Seq259.MAB39.15.barley 81
802 soybean|gbl62|BU551043 TI soybean 1371 Seq256.MAB37.15.tomato 88
803 soybean gbl 62 EV282184 TI soybean 1372 Seq371.MAB 166.15.poplar 82
804 soybean|gbl62|B1967468 TI soybean 1373 Seq3 6 8. MAB 164.15 .barley 80
805 soybean gb 162 B1321879 TI soybean 1374 Seq259.MAB39.15.barley 81
806 soybean|gbl62|AW132704 TI soybean 1375 Seq256.MAB37.15.tomato 90
807 soybean|gbl62|BU764498 TI soybean 1376 Seq256.MAB37.15.tomato 86
808 soybean|gbl62|CA953156 TI soybean 1377 Seq298.MAB 126.15.grape 80
809 soybeangb 162 CF922618 TI soybean 1378 Seq259.MAB39.15.barley 84
810 soybean|gbl62|BU544425 TI soybean 1379 Seq357.MAB157.15.sugarca ne 81
811 soybean|gbl62|BU765332 TI soybean 1380 Seq233.MAB22.15.tomato 80
812 soybean|gbl62|CA936077 TI soybean 1381 Seq376.MAB 168.15.grape 83
813 soybean|gbl62|BE823013 TI soybean 1382 Seq376.MAB 168.15.grape 83
814 soybean|gbl62|CD417415 TI soybean 1383 Seq370.MAB 165.15.grape 85
815 soybean|gbl62|BE660691 TI soybean 1384 Seq3 62. MAB 161.15 .poplar 81
816 soybean|gbl62|CD395628 TI soybean 1385 Seq370.MAB 165.15.grape 82
817 soybean|gbl62|BU549206 T2 soybean 1386 Seq259.MAB39.15.barley 80
818 soybean|gbl62|AW351120 TI soybean 1387 Seq298.MAB 126.15.grape 82
819 soybean|gbl62|AWl32704 T2 soybean 1388 Seq256.MAB37.15.tomato 90
820 soybeangb 162 BE584244 TI soybean 1389 Seq256.MAB37.15.tomato 91
821 spruce|gbl62|CO234968 TI spruce 1390 Seq344.MAB 152.15.grape 83
822 spurge|gbl61|DV146052 TI spurge 1391 Seq357.MAB157.15.sugarca ne 81
823 spurge gbl61 DV127024 TI spurge 1392 Seq344.MAB 152.15.grape 83
824 spurge gbl61 DV124157 TI spurge 1393 Seq376.MAB 168.15.grape 85
825 strawberry gbl 64 EX683450 TI strawberry 1394 Seq348.MAB154.15.sugarca ne 81
Polyn ucleoti de SEQ ID NO: Cluster name Organism eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
826 strawberry|gbl64|EX683265 T1 strawberry 1395 Seq370.MAB 165.15.grape 81
827 strawberry|gbl64|DY675409 T1 strawberry 1396 Seq256.MAB37.15.tomato 81
828 sugarcane|gb 157.2|CA115287 T1 sugarcane 1397 Seq357.MAB157.15.sugarca ne 88
829 sugarcane|gbl57.2|CA216001 T1 sugarcane 1398 Seq259.MAB39.15.barley 85
830 sugarcane gb 157.2 CA072819 T1 sugarcane 1399 Seq241.MAB28.15.rice 83
831 sugarcane|gbl57.2|CA125036 T1 sugarcane 1400 Seq291 .MAB 123.15 .barley 82
832 sugarcane|gbl57.2|CA071646 T1 sugarcane 1401 Seq286.MAB104.15.rice 90
833 sugarcane gb 157.2 CAI 17936 T2 sugarcane 1402 S eq228. MAB 19.15. sorghum 93
834 sugarcane|gbl57.2|BQ537163 T1 sugarcane 1403 Seq276.MAB49.15.maize 96
835 sugarcane|gbl57.2|CA074253 T1 sugarcane 1404 Seq241.MAB28.15.rice 83
836 sugarcane|gbl57.2|CA102030 T1 sugarcane 1405 Seq385.MAB173.15.barley 85
837 sugarcane gb 157.2 CA068084 T1 sugarcane 1406 Seq366.MAB 163.15.barley 80
838 sugarcane gb 157.2 CA233048 T1 sugarcane 1407 Seq290.MAB 122.15.maize 80
839 sugarcane gb 157.2 CA090429 T1 sugarcane 1408 Seq288.MAB121.15.sugarca ne 95
840 sugarcane gb 157.2 CA095299 T1 sugarcane 1409 Seq370.MAB 165.15.grape 80
841 sugarcane gb 157.2 BQ533298 T1 sugarcane 1410 Seq311. MAB 13 4.15 .barley 95
842 sugarcane|gbl57.2|CA107649 T1 sugarcane 1411 Seq248.MAB33.15.maize 90
843 sugarcane|gbl57.2|BQ536274 T1 sugarcane 1412 Seq231.MAB21.15.rice 88
844 sugarcane gb 157.2 CAI 17936 T1 sugarcane 1413 S eq228. MAB 19.15. sorghum 94
845 sugarcane gb 157.2 BQ533234 T1 sugarcane 1414 Seq221 .MAB 15.15. sorghum 99
846 sugarcane|gbl57.2|CA072307 T1 sugarcane 1415 Seq309.MAB 133.15.barley 93
847 sugarcane|gbl57.2|CA073476 T1 sugarcane 1416 Seq290.MAB 122.15.maize 91
848 sugarcane|gbl57.2|CA065809 T1 sugarcane 1417 Seq366.MAB 163.15.barley 80
849 sugarcane|gbl57.2|CA072307 T2 sugarcane 1418 Seq309.MAB 133.15.barley 93
850 sunflower|gbl62|DY909111 T 1 sunflower 1419 Seq336.MAB 147.15.tobacco 83
851 sunflower|gbl62|DY941035 T 1 sunflower 1420 Seq376.MAB 168.15.grape 82
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
852 sunflower gbl62 CD857487 T 1 sunflower 1421 Seq370.MAB 165.15.grape 81
853 sunflower|gbl62|DY942252 T 1 sunflower 1422 Seq311. MAB 13 4.15 .barley 80
854 sunflowergbl62 CD850784 T 1 sunflower 1423 Seq256.MAB37.15.tomato 83
855 sunflowergbl62 BQ968872 T 1 sunflower 1424 Seq357.MAB157.15.sugarca ne 83
856 sunflower|gbl62|EE616266 T 1 sunflower 1425 Seq256.MAB37.15.tomato 84
857 sunflower|gbl62|EE641694 T 1 sunflower 1426 Seq256.MAB37.15.tomato 84
858 sunflower|gbl62|DY924220 T 1 sunflower 1427 Seq259.MAB39.15.barley 81
859 sunflower|gbl62|DY910907 T 1 sunflower 1428 Seq370.MAB 165.15.grape 80
860 sunflower|gbl62|AY029172 T 1 sunflower 1429 Seq321.MAB 139.15.cotton 81
861 sunflower|gbl62|DY909077 T 1 sunflower 1430 S eq3 21. MAB 139.15.cotton 80
862 sunflower|gbl62|DY921635 T 1 sunflower 1431 Seq376.MAB 168.15.grape 83
863 sunflowergbl62DY913894 T 1 sunflower 1432 Seq256.MAB37.15.tomato 82
864 switchgrass gb 165 FE608718 T1 switchgrass 1433 Seq370.MAB 165.15.grape 81
865 switchgrass|gb 165 |FE6245 81 T1 switchgrass 1434 Seq3 3 3 .MAB 145.15 .barley 87
866 switchgrass|gbl65|FE604798 T1 switchgrass 1435 Seq269.MAB45.15. wheat 90
867 switchgrass|gbl65|DN151012 T1 switchgrass 1436 Seq309.MAB 133.15.barley 90
868 switchgrass|gb 165|FE619903 T1 switchgrass 1437 Seq383.MAB 172.15.sugarca ne 95
869 switchgrass|gbl 65 |DN 144676 T1 switchgrass 1438 Seq385.MAB173.15.barley 87
870 switchgrass|gbl65|FE609872 T1 switchgrass 1439 S eq228. MAB 19.15. sorghum 89
871 switchgrass|gbl 65 |FE617860 T1 switchgrass 1440 Seq3 81 .MAB 171.15. sugarca ne 88
872 switchgrass|gbl65|DN145750 T1 switchgrass 1441 Seq221 .MAB 15.15. sorghum 95
873 switchgrass gb 165 FE597811 T1 switchgrass 1442 Seq248.MAB33.15.maize 83
874 switchgrass|gb 165|FE647199 T1 switchgrass 1443 Seq3 81 .MAB 171.15. sugarca ne 90
875 switchgrass|gbl65|DN145034 T1 switchgrass 1444 Seq276.MAB49.15.maize 95
876 switchgrass|gb 165 |FE617335 T1 switchgrass 1445 Seq286.MAB104.15.rice 91
877 switchgrass gb 165 FE597809 T1 switchgrass 1446 Seq350.MAB 154.15.sugarca ne 95
Polyn ucleoti de SEQ ID NO: Cluster name Organism /Y/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
878 switchgrass gbl65 FE597811 T2 switchgrass 1447 Seq248.MAB33.15.maize 85
879 switchgrass|gbl65|FE635691 T1 switchgrass 1448 Seq311. MAB 13 4.15 .barley 95
880 switchgrass|gbl65|FE653022 T1 switchgrass 1449 Seq385.MAB173.15.barley 83
881 switchgrass|gb 165 |DN 144793 T1 switchgrass 1450 Seq259.MAB39.15.barley 90
882 switchgrass|gbl65|FE641674 T1 switchgrass 1451 Seq309.MAB 133.15.barley 89
883 thellungiella|gbl57.2|DN7756 06 T1 thellungiella 1452 Seq212 .MAB 10.15. arabidop sis 82
884 thellungiella |gbl57.2|DN7732 28 T1 thellungiella 1453 S eq211. MAB 9.15. arabidop si s 98
885 thellungiella|gbl57.2|DN7727 71 T1 thellungiella 1454 S eq208. MAB 7.15. arabidop si s 89
886 thellungiella gbl 57.2DN7744 22 T1 thellungiella 1455 Seq360.MAB 159.15.canola 83
887 thellungiella gb 157.2 DN7741 40 T1 thellungiella 1456 Seq284.MAB100.15.arabido psis 86
888 tobacco|gbl62|DW003503 T1 tobacco 1457 Seq329.MAB 143.15.tomato 93
889 tobacco|gbl62|BP532373 T1 tobacco 1458 Seq357.MAB157.15.sugarca ne 82
890 tobaccogbl62 CN949739 T1 tobacco 1459 Seq370.MAB 165.15.grape 84
891 tobacco gbl62BQ843111 T1 tobacco 1460 Seq319. MAB 138.15 .potato 90
892 tobacco|gbl62|EB683054 T1 tobacco 1461 Seq307.MAB131.15.tomato 89
893 tobacco gbl 62 EB428197 T1 tobacco 1462 Seq222.MAB16.15.rice 80
894 tobacco|gbl62|EB445060 T1 tobacco 1463 Seq283.MAB99.15.tomato 90
895 tobacco|gbl62|EB447202 T1 tobacco 1464 Seq3 90.MAB 175.15 .tomato 88
896 tobacco|gbl62|DW001113 T1 tobacco 1465 Seq256.MAB37.15.tomato 88
897 tobacco|gbl62|EH623692 T1 tobacco 1466 Seq303.MAB 129.15.tomato 85
898 tomato|gbl64|BG127210 T1 tomato 1467 Seq342. MAB 151.15 .potato 82
899 tomato gbl64 BG128089 T2 tomato 1468 Seq222.MAB16.15.rice 80
900 tomato|gbl64|AW219181 T1 tomato 1469 Seq256.MAB37.15.tomato 90
901 tomato|gbl64|BG127288 T1 tomato 1470 Seq370.MAB 165.15.grape 83
902 tomato|gbl64|BG133509 T1 tomato 1471 Seq256.MAB37.15.tomato 88
903 tomato gbl64 BG131241 T1 tomato 1472 Seq309.MAB 133.15.barley 80
904 tomato|gbl64|BG129621_Tl tomato 1473 Seq350.MAB154.15.sugarca ne 80
905 tomato|gbl64|A1779004 T1 tomato 1474 Seq309.MAB 133.15.barley 81
906 tomato|gbl64|BG129572 T1 tomato 1475 Seq321.MAB 139.15.cotton 80
907 tomato|gbl64|BG135408 T1 tomato 1476 Seq319. MAB 138.15 .potato 98
908 triphysaria gbl64DRl 73028 T1 triphysaria 1477 Seq329.MAB 143.15.tomato 81
909 triphysaria|gb 164|BM3 57524 T2 triphysaria 1478 Seq283.MAB99.15.tomato 85
910 triphysaria gb 164 E Y13 3 83 8 T1 triphysaria 1479 Seq311. MAB 13 4.15 .barley 80
911 triphysaria|gbl64|BM357406 T1 triphysaria 1480 Seq329.MAB 143.15.tomato 83
912 triphysaria|gbl64|BM357011 T1 triphysaria 1481 Seq259.MAB39.15.barley 80
Polyn ucleoti de SEQ ID NO: Cluster name Organism Po/j’P eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
913 triphysaria|gb 164|BM3 57524 T1 triphysaria 1482 Seq376.MAB 168.15.grape 85
914 triphysaria|gb 164 |E Y137290 T1 triphysaria 1483 Seq256.MAB37.15.tomato 88
915 wheat|gbl64|CA484259 T1 wheat 1484 Seq241.MAB28.15.rice 84
916 wheat|gbl64|BE606422 T1 wheat 1485 Seq379.MAB170.15.barley 96
917 wheat|gbl64|BE406378 T1 wheat 1486 Seq219.MAB14.15.rice 80
918 wheat gb 164 BE470780 T1 wheat 1487 Seq221 .MAB 15.15. sorghum 84
919 wheat gb 164 BE418087 T1 wheat 1488 Seq325.MAB141.15.barley 95
920 wheat|gbl64|BQ294643 T1 wheat 1489 Seq269.MAB45.15. wheat 94
921 wheat|gbl64|BE415314 T1 wheat 1490 Seq250.MAB34.15.barley 82
922 wheat|gbl64|AL822647 T1 wheat 1491 Seq259.MAB39.15.barley 98
923 wheat|gbl64|BE406667 T1 wheat 1492 Seq250.MAB34.15.barley 89
924 wheat|gbl64|BF475039 T1 wheat 1493 Seq221 .MAB 15.15. sorghum 83
925 wheatgbl64 CK196180 T1 wheat 1494 Seq323.MAB 140.15.barley 80
926 wheat|gbl64|BE403745 T1 wheat 1495 Seq379.MAB170.15.barley 97
927 wheat|gbl64|BQ620260 T1 wheat 1496 Seq311. MAB 13 4.15 .barley 100
928 wheat|gbl64|BM138204 T1 wheat 1497 Seq3 3 3 .MAB 145.15 .barley 91
929 wheat|gbl64|BE401114 T1 wheat 1498 Seq291 .MAB 123.15 .barley 94
930 wheat|gbl64|BE498161 T1 wheat 1499 Seq3 88.MAB 174.15.barley 93
931 wheat|gbl64|BQ744502 T1 wheat 1500 Seq250.MAB34.15.barley 85
932 wheat|gbl64|BE415172 T1 wheat 1501 Seq366.MAB 163.15.barley 94
933 wheat|gbl64|CD490875 T1 wheat 1502 Seq276.MAB49.15.maize 97
934 wheat gbl 64 CA625741 T1 wheat 1503 Seq309.MAB 133.15.barley 87
935 wheat|gbl64|BE443720 T1 wheat 1504 Seq318.MAB 137.15.barley 94
936 wheat|gbl64|BE420294 T1 wheat 1505 Seq290.MAB 122.15.maize 84
937 wheat|gbl64|BE516581 T1 wheat 1506 Seq387.MAB174.15.barley 95
938 wheat|gbl64|BE406039 T1 wheat 1507 Seq3 3 3 .MAB 145.15 .barley 90
939 wheat|gbl64|BM136483 T1 wheat 1508 Seq3 3 3 .MAB 145.15 .barley 92
940 wheat|gbl64|BE425976 T1 wheat 1509 Seq250.MAB34.15.barley 81
941 wheatgbl64 CN011148 T1 wheat 1510 Seq270.MAB45.15. wheat 84
942 wheat|gbl64|BE419039 T1 wheat 1511 Seq250.MAB34.15.barley 80
943 wheat|gbl64|CA603413 T1 wheat 1512 Seq323.MAB 140.15.barley 85
944 wheatgbl64CA743309 T1 wheat 1513 Seq321. MAB 139.15. cotton 80
945 wheatgbl64 BG262336 T1 wheat 1514 Seq366.MAB 163.15.barley 94
946 wheatgbl64 CD881765 T1 wheat 1515 Seq219.MAB14.15.rice 80
947 wheat|gbl 64 |BE3 52629 T1 wheat 1516 Seq291 .MAB 123.15 .barley 96
948 wheat|gbl64|BE398656 T1 wheat 1517 Seq308.MAB 132.15.barley 97
949 wheat|gbl64|BE403195 T1 wheat 1518 Seq291 .MAB 123.15 .barley 94
950 wheatgbl64 BE488904 T1 wheat 1519 Seq367.MAB 163.15.barley 91
951 wheat gb 164 BE492528 T1 wheat 1520 Seq311. MAB 13 4.15 .barley 100
952 wheat gbl64 BE427383 T1 wheat 1521 Seq219.MAB14.15.rice 80
953 wheat|gbl64|CA646957 T1 wheat 1522 Seq250.MAB34.15.barley 89
954 wheat|gbl64|BE443720 T2 wheat 1523 Seq318.MAB 137.15.barley 92
955 wheat gb 164 BE490408 T1 wheat 1524 Seq264.MAB42.10.sorghum 81
956 wheat|gbl64|BE420295 T1 wheat 1525 Seq379.MAB170.15.barley 96
957 wheat gb 164 AL825998 T1 wheat 1526 Seq308.MAB 132.15.barley 97
958 wheat|gbl64|CA693465 T1 wheat 1527 Seq308.MAB 132.15.barley 97
959 wheat|gbl64|BE585772 T1 wheat 1528 Seq366.MAB 163.15.barley 95
960 wheat gb 164|CA613 914_T 1 wheat 1529 Seq356.MAB157.15.sugarca ne 84
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Polyn ucleoti de SEQ ID NO: Cluster name Organism Polyp eptid e SEQ ID NO: Homolog to a polypeptide encoded by polynucleotide SEQ ID NO. % Glob al identi ty
1656 >tomato|gbl64|BG129621 T 1 tomato 1660 Seql649. MAB66.tomato 82
1657 potato|gbl57.2|BE921143_Tl potato 1661 Seql649. MAB66.tomato 82
1658 pepper|gbl57.2|BM061807 T 1 pepper 1662 Seql649. MAB66.tomato 80
1659 >triphysaria|gb 164|BM35701 1_T1 triphysaria 1663 Seql649. MAB66.tomato 80
Table 2: *- Homology was calculated as % of identity over the aligned sequences. The query sequences were polynucleotide sequences SEQ ID NOs:l, 3, 5, 7, 9, 10, 11, 13, 15, 16, 17, 19, 21, 23, 25, 26, 28, 29, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55, 57, 59, 61, 63, 65, 67, 69, 71 ,73 ,75 ,77, 79, 81, 82, 84, 86, 88, 90, 91, 93, 94, 96, 98, 100, 101, 103, 105, 107, 109, 111, 113, 115, 116, 118, 119, 121, 122, 124, 126, 128, 130, 132, 134, 135, 138, 140, 142, 143, 145, 147, 149, 151, 153, 155, 157, 161, 163, 165, 168, 169, 170, 171, 173, 175, 177, 179, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 and 1649, and the subject sequences are protein sequences identified in the database based on greater than 80 % identity to the predicted translated sequences of the query nucleotide sequences. Shown are the homologous polypeptides and the polynucleotides encoding same.
EXAMPLE 2
GENERA TING THE PUTA TIVE ABST GENES
Several DNA sequences of the ABST genes are synthesized by GeneArt (Hypertext Transfer Protocol://World Wide Web (dot) geneart (dot) com/). Synthetic DNA is designed in silico, based on the encoded amino-acid sequences of the ABST genes and using codon-usage Tables calculated from plant transcrip tomes (example of such Tables can be found in the Codon Usage Database available online at Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/). The optimized coding sequences are designed in a way that no changes are introduced in the encoded amino acid sequence while using codons preferred for expression in dicotyledonous plants (mainly tomato and Arabidopsis) and monocotyledonous plants such as maize. At least one silent mutation per 20 nucleotide base pairs is introduced in the sequence compared to the original sequences to avoid possible silencing when overexpressing the gene in the target crop. To the optimized sequences the following restriction enzymes sites are added- Sall, Xbal, BamBI, Smal at the 5' end and Sad at the 3' end. The sequences synthesized by the supplier (GeneArt, Gmbh) are cloned in the pCR-Script plasmid.
2017228711 15 Sep 2017
EXAMPLE 3
GENE CLONING AND GENERATION OF BINARY VECTORS FOR PLANT EXPRESSION
To validate their role in improving ABST and yield, selected genes were overexpressed in plants, as follows.
Cloning strategy
Selected genes from those presented in Example 1 were cloned into binary vectors for the generation of transgenic plants. For cloning, the full-length open reading frames (ORFs) were identified. EST clusters and in some cases mRNA sequences were analyzed to identify the entire open reading frame by comparing the results of several translation algorithms to known proteins from other plant species.
In order to clone the full-length cDNAs, reverse transcription (RT) followed by polymerase chain reaction (PCR; RT-PCR) was performed on total RNA extracted from leaves, roots or other plant tissues, growing under either normal or nutrient deficient conditions. Total RNA extraction, production of cDNA and PCR amplification was performed using standard protocols described elsewhere (Sambrook J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory Press, New York.) which are well known to those skilled in the art. PCR products were purified using PCR purification kit (Qiagen)
Usually, 2 sets of primers were prepared for the amplification of each gene, via nested PCR (meaning first amplifying the gene using external primers and then using the produced PCR product as a template for a second PCR reaction, where the internal set of primers are used). Alternatively, one or two of the internal primers were used for gene amplification, both in the first and the second PCR reactions (meaning only 2-3 primers were designed for a gene). To facilitate further cloning of the cDNAs, an 8-12 bp extension is added to the 5' of each internal primer. The primer extension includes an endonuclease restriction site. The restriction sites are selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) the restriction sites in the forward and reverse primers are designed such that the digested cDNA is inserted in the sense direction into the binary vector utilized for transformation. In Table 3 below, primers used for cloning ABST genes are provided.
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Table 3
Cloned ABST genes from cDNA libraries or genomic DNA and the primers used for the cloning
Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
ΜΑΒΙ 1530 201 EcoRV ΜΑΒΙ EF EcoRV AAGATATCAGACCAGAGGAGA AGACTCGATC (SEQ ID NO: 1567) ΜΑΒΙ NF EcoRV AAGATATCAGACTCCGTTCGGA GAAAAGG (SEQ ID NO: 1568) ΜΑΒΙ ER EcoRV ATGATATCTGAAGAACATCGCC TTGTCATC (SEQ ID NO: 1569) ΜΑΒΙ NR EcoRV AAGATATCACCTTGTCATCGGA TCATCTCC (SEQ ID NO: 1570)
MAB1GA (optimized for expression in Maize and G.Max) 1531 Synthetic product (from pGA14_MABl_GA)
MAB 14 1538 219 EcoRV MAB 14 EF EcoRV ATGATATCCAACGAATGAAGA CTAGTAGCTG (SEQ ID NO: 1571) MAB 14 NF EcoRV ATGATATCCCAGATGGAATCCT GCCCT (SEQ ID NO: 1572) MAB 14 ER EcoRV ATGATATCGTGTCAATGAAGG GAACGTGC (SEQ ID NO: 1573) MAB 14 NR EcoRV ATGATATCGCAAATGGATTCAG ATATTCTG (SEQ ID NO: 1574)
MAB14GA (optimized for expression in Maize) 1539 Synthetic product ( from pGA14_MAB14_GA)
MAB 10 1532 212 Sall, Xbal MAB 10 F Sal GCAGTCGACAACTCACAGTTCC AAACACACA (SEQ ID NO: 1575) MAB lOExtRXbaGGTCTAGAATGTAAATGTCTTC GTATTAGGC (SEQ ID NO: 1576) MAB lONRXbaCCTCTAGAATCACCCGAAATAA CTAGTGTC (SEQ ID NO: 1577)
MAB10GA (optimized for expression in Maize) 1533 Synthetic product ( from pGA18_MAB10_GA)
2017228711 15 Sep 2017
Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB25 1549 237 Pstl, Smal MAB25 EF Pstl- AACTGCAGCCATCGTCGTAATC CTTCTAGC (SEQ ID NO:1578) MAB25 NF PstlAACTGCAGTAATCATGGGGAG GAAATCTC (SEQ ID NO:1579) MAB25 ER SmalGGGTGACAATTCCGAGTCTCAG C (SEQ ID NO: 1580) MAB25 NR SmalTCCCGGGCAATTGGTCAATGGC ACTC (SEQ ID NO:1581)
MAB25GA (optimized for expression in Maize) 1550 Synthetic product ( from pGA14_MAB25_GA)
MAB 134 1665 311 Sall, Xbal MAB 134 EF Sall- AATGTCGACTCTCGTCTTGCTC CCAGAG (SEQ ID NO: 1582) MAB 134 NF Sall- AATGTCGACCGACACCCTTCTC CTCCTC (SEQ ID NO: 1583) MAB 134 ER Xbal- TTTCTAGAATCATATTCCAACA TCCACTTC (SEQ ID NO: 1584) MAB 134 NR Xbal- TTTCTAGACTGCTATGTTCCAC TGACTACAC (SEQ ID NO: 1585)
MAB99 1566 283 Sall, Sacl MAB99 NF Sall- AAAGTCGACCAGTTAATTCTCC GTTGTCTACTC (SEQ ID NO:1586) MAB99 NR Sacl- TGAGCTCCTGCTTGAAACTTGC TGCTAG (SEQ ID NO: 1587)
MAB36 1554 254 Sall, Xbal MAB 36 F Sal GGAGTCGACACAGAAATGGGT GGTTTGAAG (SEQ ID NO: 1588) MAB 36 Ext R Xba CCTCTAGAAATGATCACTCACT GCAACTTAG (SEQ ID NO:1589) MAB 36 NRXbaCCTCTAGACACTCACTGCAACT TAGAAACATC (SEQ ID NO: 1590)
2017228711 15 Sep 2017
Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB7 1563 208 Sall, Xbal MAB 7 Ex F Sal AACGTCGACGCTCATTTCTCTT CTTCTTTGG (SEQ ID NO:1591) MAB 7 NF Sal GACGTCGACTCTTCTTTGGTTC TTACATTTCTC (SEQ ID NO:1592) MAB 7ExRXbaTCTCTAGAGCAAGACGTTATAA ACCATGC (SEQ ID NO: 1593) MAB 7 NR Xba - TCTCTAGAAGAAGACACGCTG GACAATG (SEQ ID NO:1594)
MAB44 1557 267 Sall, Sacl MAB 44 NF sal AAGGTCGACCATAAAGAACAG TGACAGGCG (SEQ ID NO: 1595) MAB 44 NR Sc AGAGCTCCACGTAGTACATTTT CACAGCAC (SEQ ID NO: 1596)
MAB44GA (optimized for expression in Maize) 1558 Synthetic product (from pCR4BluntTOPOMAB44GA)
MAB6 1561 207 Sall, Xbal MAB 6 - Ex F Sal ACCGTCGACCCTTCTCCAATTT CGTAAGC (SEQ ID NO: 1597) MAB 6 NF Sal ACCGTCGACTTCGTAAGCTCAA AGATTTCG (SEQ ID NO:1598) MAB 6 - Ext R Xbal CCTCTAGAACGACTTTTAATCC CTCCAAC (SEQ ID NO:1599) MAB 6 - NR Xbal CCTCTAGACTCCAACAGCCACT ACAACC (SEQ ID NO: 1600)
MAB6GA (optimized for expression in Maize) 1562 Synthetic product (from pGA15_MAB6_GA)
MAB9 1564 211 EcoRV MAB9 F EcoRV AAGATATCGGTTGCTGAGGAA TCGAAGTAG (SEQ ID NO: 1601) MAB9 ER EcoRV TTGATATCGAGCCAAGTCACAA GGAGTTTAC (SEO ID NO :1602) MAB9 NR EcoRV TTGATATCCTCCGAGTGTCGCA GTAAGC (SEQ ID NO:1603)
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Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB9GA (optimized for expression in Maize and G.Max) 1565 Synthetic product (from pGA15_MAB9_GA)
MAB 100 1534 284 Sall, Xbal MAB 100 EF Sall- AATGTCGACCCAAGTTAAACTT CATATCATACAC (SEQ ID NO: 1604) MAB 100 NF Sall- AATGTCGACGAAGAGTTATTAT GGCGAGCT (SEQ ID NO :1605) MAB 100 ER Xbal- AATGTCGACCCAAGTTAAACTT CATATCATACAC (SEQ ID NO: 1606) MAB 100 NR Xbal- AATCTAGACAAACCCAACTTAT TACATTACG (SEQ ID NO: 1607)
MAB13 1536 217 Sacl, Sall MAB 13 F Sall new AATGTCGACCTCGAAAATGGC CACCATTAG (SEQ ID NO: 1608) MAB 13 ExR Sc CGAGCTCCAAAAATGCAAGAA TCAAGAG (SEQ ID NO: 1609) MAB 13 F Sal AAGGTCGACTTCTCTCCAAAAT GGCCAC (SEQ ID NO: 1610) MAB 13 NR Sc TGAGCTCTGCAAGAATCAAGA GAAATTTG (SEQ ID NO: 1611)
MAB32 1552 247 EcoRV MAB32 F EcoRV- AAGATATCCTCCACTTGTTGTT CAATTCCC (SEQ ID NO: 1612) MAB32 ER EcoRVATGATATCGATCTGAACAGCA GTAAGTAAGCC (SEQ ID NO:1613) MAB32 NR EcoRV- ATGATATCTAAGAAGAACAAG ACATGGATCG (SEQ ID NO: 1614)
MAB35 1553 252 Smal MAB35 FCGTGAGAACTAAGAAACACCC (SEQ ID NO:1615) MAB35 ER SmalTCCCGGGACATCTTTTCAACTA AACCAAGAC (SEQ ID NO: 1616) MAB35 NR SmalTCCCGGGCTAAACCAAGACTTA CACAAGACG (SEQ ID NO: 1617)
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Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB 146 1666 334 Sall, Xbal MAB 146 F Sal- ATTGTCGACAGAGTTATGGGA GATAATAGAGGA (SEQ ID NO:1618) MAB 146 ER Xba - ATTCTAGACTCATTCTGAGCTT TACATGTTC (SEQ ID NO: 1619) MAB 146 NR Xba- TTTCTAGATTGGTTTACACCTC AACTCACTAC (SEQ ID NO: 1620)
MAB2 1547 Non coding Sall, Xbal MAB2 F Sall AATGTCGACAACAAATGATCCT TCAGGCAGTTAAAG (SEQ ID NO:1621) MAB2 R Xba TTTCTAGATATTAAAACTTAGA TTCGGGATCAG (SEQ ID NO: 1622)
MAB20 1548 229 Pstl, Smal MAB20 EF PstlAACTGCAGGATCATCACTTCTC AGATTTCG (SEQ ID NO: 1623) MAB20 NF PstlAACTGCAGAAAAATGAATTCA GAATCGCTAG (SEQ ID NO: 1624) MAB20 ER SmalAACTGCAGGATCATCACTTCTC AGATTTCG (SEQ ID NO: 1625) MAB20 NR SmalTCCCGGGCAATCTGACCTCAAA ACTCCC (SEQ ID NO :1626)
MAB43 1556 265 Pstl, Smal MAB43 NF Pstl AACTGCAGGATCAATGAAGAT TCGGAACAG (SEQ ID NO: 1627) MAB43 ER Smal TCCCGGGTACAACAAGAAACC TCTGATTC (SEQ ID NO: 1628) MAB43 NR Smal TCCCGGGCCTGTGCCACAGCTA TACTTAC (SEQ ID NO :1629)
MAB46 1559 271 Sall, Sacl MAB 46 ExF Sal - GAAGTCGACATCCGTAGTTTCA GTTTCGTCC (SEQ ID NO:1630) MAB 46 NF Sal - GAAGTCGACCTTGTCTGTTCCA GATGAAATTG (SEQ ID NO :1631) MAB46 ExR Sc - TGAGCTCCTCTATCGACGTCCG GATTC (SEQ ID NO: 1632) MAB 46 NR Sc - TGAGCTCCGTCCGGATTCATAA ACAAC (SEQ ID NO: 1633)
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Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB50 1560 277 Smal MAB 50 ExF Sal GGAGTCGACCATCGGGACACA TCTTTAGG (SEQ ID NO:1634) MAB50 NF CATCTTTAGGCTCAAGGATTC (SEQ ID NO: 1635) MAB50 ExR Sac TGAGCTCGATCCTCGTTTATTA CAAGTCTG (SEQ ID NO: 1636 ) MAB50 NR Sma TCCCGGGCACACCAAGATTGAT TACAAAGAG (SEQ ID NO: 1637)
MAB66 1654 1655 Sall, Xbal MAB66 F SalAATGTCGACGATTGGAGATAG GCAGGCA (SEQ ID NO :163 8) MAB66 ER Xba TTTCTAGAGGTAGCCAAAGCTG ACACTC (SEQ ID NO: 1639) MAB66 NR Xba AATCTAGAGAGGCATATGCAC TTCTTATCG (SEQ ID NO: 1640)
MAB4 1555 205 EcoRV MAB4 EF EcoRV- AAGATATCCAGGACGGGTTCTC GATCAG (SEQ ID NO: 1641) MAB4 NF EcoRVAAGATATCCAGCGAACACGTC TACGATG (SEQ ID NO :1642) MAB4 ER EcoRV - ATGATATCGCACGAGTTCAACT CAGCTG (SEQ ID NO:1643) MAB4 NR EcoRV- ATGATATCGAACTGCTTGAGAT GTAACAGCT (SEQ ID NO :1644)
MAB15GA (optimized for expression in Arabidopsis and maize) 1541 221 Xbal, Sacl Synthetic product (from pGA4_MAB15)
MAB15a_G A (optimized for expression in Maize) 1667 Synthetic product (from pGA18 MAB15a_GA)
MAB15GA original (original sequence, not optimize) 1540 Synthetic product (from pGAl 4MAB15 _(E VO220)original)
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Gene Id Polynucleotide SEQ ID NO. of the cloned gene Polypeptide SEQ ID NO. of the encoded polypeptide Restriction Enzymes used for cloning Primers used for amplification (SEQ ID NO:)
MAB17GA (optimized for expression in Arabidopsis and maize) 1542 224 Xbal, Sacl Synthetic product (from pGA4_MAB17)
MAB17a_G A (optimized for expression in Maize) 1544 Synthetic product (from pCR4BluntTOPO_MAB17a_GA)
MAB17GA original (original sequence, not optimize) 1543 Synthetic product (pGA14_MAB17_(EVO222)original)
MAB137G A (optimized for expression in Maize, Arabidopsis and tomato) 1537 317 Xbal, Sacl Synthetic product (from pGA15_MAB137)
MAB3GA (optimized for expression in Maize, Arabidopsis and tomato) 1551 203 Xbal, Sacl Synthetic product (from pCR4Blunt- Topo_MAB3)
MAB3_GA_ original (original sequence, not optimize) 1668 Synthetic product (from pGA 14_MAB3_(E VO23 5)-original)
MAB18GA (optimized for expression in Arabidopsis and maize) 1545 225 Xbal, Sacl Synthetic product (from pGA4_MAB18)
Control Gene: GUI 1664
Table 3. Presented are the cloned AB ST genes and control gene(s) by the Gene Id number and the polynucleotide SEQ ID NO. Also presented are the primers and the restriction enzymes used to clone the AB ST genes.
PCR products were digested with the restriction endonucleases (Roche, Switzerland) according to the sites design in the primers (Table 3). Each digested PCR product was inserted into a high copy vector originated from pBlue-script KS plasmid
2017228711 15 Sep 2017 vector (pBlue-script KS plasmid vector, Hypertext Transfer Protocol://World Wide Web (dot) stratagene (dot) com/manuals/212205 (dot) pdf). In case of the high copy vector originated from pBlue-script KS plasmid vector (pGN) PCR product was inserted in the high copy plasmid upstream to the NOS terminator (SEQ ID NO: 1651) originated from pBI 101.3 binary vector (GenBank Accession No. U12640, nucleotides 4417 to 4693), Table 4 below. In other cases (pKSJ_6669a) the At6669 promoter (SEQ ID NO: 1652) is already cloned into the pBlue-script KS, so the gene is introduced downstream of the promoter (Table 4 below).
Sequencing of the inserted genes was performed, using the ABI 377 sequencer (Applied Biosystems). In some cases, after confirming the sequences of the cloned genes, the cloned cDNA accompanied with the NOS terminator was introduced into the binary vectors pGI containing the At6669 promoter via digestion with appropriate restriction endonucleases. In other cases the cloned cDNA accompanied with the At6669 promoter was introduced into the pGI vector (that hasn't already contained the At6669 promoter). In any case the insert was followed by single copy of the NOS terminator (SEQ ID NO: 1651). The digested products and the linearized plasmid vector were ligated using T4 DNA ligase enzyme (Roche, Switzerland).
Table 4
Genes cloned from cDNA libraries or genomic DNA in a High copy plasmid
Gene Name High copy Plasmid Amplified from
ΜΑΒΙ pKSJ 6669 RNA
ΜΑΒΙ Gene Art
MAB 10 Gene Art
MAB 10 pGN RNA
MAB 14 pKSJ 6669 RNA
MAB 14 Gene Art
MAB15 pGN Gene Art (3 plasmids)
MAB17 pGN Gene Art (3 plasmids)
MAB 137 pGN Gene Art
MAB25 pKSJ 6669 RNA
MAB25 Gene Art
MAB3 pGN Gene Art (2 plasmids)
MAB44 pGN RNA
MAB44 Gene Art
MAB6 pGN RNA
MAB6 Gene Art
MAB9 pKSJ 6669 RNA
MAB9 Gene Art
MAB 100 pGN RNA
MAB13 pGN RNA
MAB 134 pGN RNA
MAB18 pGN Gene Art
MAB2 pGN RNA
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Gene Name High copy Plasmid Amplified from
MAB20 pKSJ 6669 RNA
MAB 146 pGN RNA
MAB32 pKSJ 6669 RNA
MAB35 pKSJ 6669 RNA
MAB36 pGN RNA
MAB43 pKSJ 6669 RNA
MAB46 pGN RNA
MAB50 pKSJ 6669 RNA
MAB7 pGN RNA
MAB99 pGN RNA
MAB66 pGN RNA
MAB4 pKSJ 6669 RNA
Table 4
The pPI plasmid vector was constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, GenBank Accession No. U47295; nucleotides 4658-4811) into the Hindlll restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession No. U12640). pGI (Figure 1) is similar to pPI, but the original gene in the back bone is GUS-Intron, rather than GUS.
At6669, the Arabidopsis thaliana promoter sequence (set forth in SEQ ID NO: 1652) is inserted in the pPI binary vector, upstream to the cloned genes by using the restriction enzymes EiindAAA and Sal\ or BamAAl (Roche), following by DNA ligation and binary plasmid extraction from positive E. coli colonies, as described above.
Positive colonies were identified by PCR using primers which were designed to span the introduced promoter (At6669) and the cloned gene in the binary vector. In all cases the forward PCR primer was the primer set forth in SEQ ID NO: 1650 (from the At6669 promoter) and the reverse primer (derived from the specific cloned gene) was as follows: For ΜΑΒΙ, the reverse primer was SEQ ID NO:1570; for MAB14, the reverse primer was SEQ ID NO: 1574; for MAB10, the reverse primer was SEQ ID NO: 1577; for MAB25, the reverse primer was SEQ ID NO:1581; for MAB134, the reverse primer was SEQ ID NO: 1585; for MAB99, the reverse primer was SEQ ID NO: 1587; for MAB36, the reverse primer was SEQ ID NO: 1590; for MAB7, the reverse primer was SEQ ID NO: 1594; for MAB44, the reverse primer was SEQ ID NO: 1596; for MAB4, the reverse primer was SEQ ID NO: 1600; for MAB9, the reverse primer was SEQ ID NO: 1603 (MAB9); for MAB100, the reverse primer was SEQ ID NO: 1606; for MAB13, the reverse primer was SEQ ID NO: 1611; for MAB32, the reverse primer was SEQ ID NO: 1614; for MAB35, the reverse primer was SEQ ID NO: 1617; for MAB146, the reverse primer was SEQ ID NO: 1620; for MAB2, the reverse primer was SEQ ID
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NO: 1622; for MAB20, the reverse primer was SEQ ID NO: 1626; for MAB43, the reverse primer was SEQ ID NO: 1629; for MAB46, the reverse primer was SEQ ID NO: 1633; for MAB50, the reverse primer was SEQ ID NO: 1637; for MAB66, the reverse primer was SEQ ID NO: 1640; for MAB4, the reverse primer was SEQ ID NO: 1644; for MAB15 synthetic gene, the reverse primer was SEQ ID NO: 1645; for MAB 17 synthetic gene, the reverse primer was SEQ ID NO: 1646; for MAB 18 synthetic gene, the reverse primer was SEQ ID NO:1647; for MAB137 synthetic gene, the reverse primer was SEQ ID NO: 1648; and for MAB3 synthetic gene, the reverse primer was SEQ ID NO: 1649, which are designed to span the introduced promoter and gene, in the binary vector.
Synthetic sequences [such as of MAB14, nucleotide SEQ ID NO:23, which encodes protein SEQ ID NO:219) of some of the cloned polynucleotides were ordered from a commercial supplier (GeneArt, GmbH). To optimize the coding sequence, codon-usage Tables calculated from plant transcriptomes were used [example of such Tables can be found in the Codon Usage Database available online at Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/]. The optimized coding sequences were designed in a way that no changes were introduced in the encoded amino acid sequence while using codons preferred for expression in dicotyledonous plants mainly tomato and Arabidopsis; and monocotyledonous plants such as maize. Such optimized sequences promote better translation rate and therefore higher protein expression levels. Parts of the sequences were ordered as the original sequences. To the optimized/non-optimized sequences flanking additional unique restriction enzymes sites were added to facilitate cloning genes in binary vectors.
Promoters used: Arabidopsis At6669 promoter (SEQ ID NO: 1652; which is SEQ ID NO:61 of W004081173 to Evogene Ltd.).
The sequences of the cloned cDNAs are provided in SEQ ID NOs: 1530-1534, 1536-1545, 1547-1566, 1654, 1665, 1666, 1667 and 1668. The protein translation of the amplified cDNA sequence matched exactly that of the initial bioinformatics prediction of the protein sequences. The predicted polypeptide sequences of the cloned polynucleotides are provided in SEQ ID NOs:201, 212, 284, 213, 217, 317, 219, 221, 224, 225, 226, 227, 229, 237, 203, 247, 252, 205, 265, 267, 271, 277, 207, 208, 211, 283, 1655, 311,334, and 254.
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EXAMPLE 4
TRANSFORMING AGROBACTERIUM TUMEFACIENS CELLS WITH BINARY VECTORS HARBORING PUTATIVE ABST GENES
Each of the binary vectors described in Example 3 above are used to transform Agrobacterium cells. Two additional binary constructs, having a GUS/Luciferase reporter gene replacing the ABST gene (positioned downstream of the At6669 promoter), are used as negative controls.
The binary vectors are introduced to Agrobacterium tumefaciens GV301, or LB4404 competent cells (about 109 cells/mL) by electroporation. The electroporation is performed using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). The treated cells are cultured in LB liquid medium at 28 °C for 3 hours, then plated over LB agar supplemented with gentamycin (50 mg/L; for Agrobacterium strains GV301) or streptomycin (300 mg/L; for Agrobacterium strain LB4404) and kanamycin (50 mg/L) at 28 °C for 48 hours. Abrobacterium colonies which developed on the selective media were analyzed by PCR using the primers described above (Example 3) with respect to identification of positive binary vector colonies. The resulting PCR products are isolated and sequenced as described in Example 3 above, to verify that the correct ABST sequences are properly introduced to the Agrobacterium cells.
EXAMPLE 5
TRANSFORMATION OF ARABIDOPSIS THALIANA PLANTS WITH PUTATIVE ABSTGENES
Arabidopsis thaliana Columbia plants (To plants) are transformed using the Floral Dip procedure described by Clough and Bent (10) and by Desfeux et al. (11), with minor modifications. Briefly, To Plants are sown in 250 ml pots filled with wet peatbased growth mix. The pots are covered with aluminum foil and a plastic dome, kept at 4 °C for 3¾ days, then uncovered and incubated in a growth chamber at 18-24 °C under 16/8 hour light/dark cycles. The To plants are ready for transformation six days before anthesis.
Single colonies of Agrobacterium carrying the binary constructs, are generated as described in Example 4 above. Colonies are cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures are incubated at 28 C
2017228711 15 Sep 2017 for 48 hours under vigorous shaking and then centrifuged at 4000 rpm for 5 minutes. The pellets comprising the Agrobacterium cells are re-suspended in a transformation medium containing half-strength (2.15 g/L) Murashige-Skoog (Duchefa); 0.044 μΜ benzylamino purine (Sigma); 112 pg/L B5 Gambourg vitamins (Sigma); 5 % sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double-distilled water, at pH of 5.7.
Transformation of To plants is performed by inverting each plant into an Agrobacterium suspension, such that the above ground plant tissue is submerged for 3-5 seconds. Each inoculated To plant is immediately placed in a plastic tray, then covered with clear plastic dome to maintain humidity and is kept in the dark at room temperature for 18 hours, to facilitate infection and transformation. Transformed (transgenic) plants are then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic To plants are grown in the greenhouse for 3-5 weeks until siliques are brown and dry. Seeds are harvested from plants and kept at room temperature until sowing.
For generating Ti and T2 transgenic plants harboring the genes, seeds collected from transgenic To plants are surface-sterilized by soaking in 70 % ethanol for 1 minute, followed by soaking in 5 % sodium hypochloride and 0.05 % triton for 5 minutes. The surface-sterilized seeds are thoroughly washed in sterile distilled water then placed on culture plates containing half-strength Murashige-Skoog (Duchefa); 2 % sucrose; 0.8 % plant agar; 50 mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates are incubated at 4 °C for 48 hours then transferred to a growth room at 25 °C for an additional week of incubation. Vital Ti Arabidopsis plants are transferred to a fresh culture plates for another week of incubation. Following incubation the Ti plants are removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants are allowed to grow in a greenhouse to maturity. Seeds harvested from Ti plants are cultured and grown to maturity as T2 plants under the same conditions as used for culturing and growing the Ti plants.
EXAMPLE 6
IMPRO VED ABST IN TISSUE CUL TURE ASS A Y
Assay 1: plant growth under Osmotic stress (PEG) in Tissue culture conditions - Osmotic stress (PEG) - conditions resembling the high osmolarity found during drought (e.g., 25 % PEG8000). One of the consequences of drought is the induction of
2017228711 15 Sep 2017 osmotic stress in the area surrounding the roots; therefore, in many scientific studies, PEG serves to simulate drought.
Surface sterilized seeds are sown in basal media [50 % Murashige-Skoog medium (MS) supplemented with 0.8 % plant agar as solidifying agent] in the presence of Kanamycin (for selecting only transgenic plants). After sowing, plates are transferred for 2-3 days at 4 °C for stratification and then grown at 25 °C under 12-hour light 12hour dark daily cycles for 7 to 10 days. At this time point, seedlings randomly chosen are carefully transferred to plates hold 25 % PEG in 0.5 MS media or normal conditions (0.5 MS media). Each plate contains 5 seedlings of same event, and 3-4 different plates (replicates) for each event. For each polynucleotide of the invention at least four independent transformation events are analyzed from each construct. Plants expressing the polynucleotides of the invention are compared to the average measurement of the control plants Mock- transgenic plants expressing the uidA reporter gene (GUS Intron GUI) under the same promoter were used as control.
Digital imaging - A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which included 4 light units (4 x 150 Watts light bulb) and located in a darkroom, was used for capturing images of plantlets sawn in square agar plates.
The image capturing process was repeated every 7 days starting at day 0 till day 14. The same camera attached with a 24 mm focal length lens (Canon EF series), placed in a custom made iron mount was used for capturing images.
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37 (Java based image processing program which was developed at the U.S National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/). Images were captured in resolution of 6 Mega Pixels (3072 x 2048 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Seedling analysis - Using the digital analysis seedling data was calculated, including leaf area, root coverage and root length.
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The Relative Growth Rate (RGR) was calculated according to the following formula I.
Formula I:
Relative growth area rate = (Δ Area / At) * (1/ Area tO)
At is the current analyzed image day subtracted from the initial day (t-tO). Thus, the relative growth area rate is in units of 1/day and length growth rate is in units of 1/day.
At the end of the experiment, plantlets were removed from the media and weighed for the determination of plant fresh weight. Relative Growth Rate is determined 10 by comparing the leaf area, root length and root coverage between each couple of sequential photographs, and results are used to resolve the effect of the gene introduced on plant vigor, under osmotic stress, as well as under optimal conditions. Similarly, the effect of the gene introduced on biomass accumulation, under osmotic stress as well as under optimal conditions, is determined by comparing the plants' fresh weight to control 15 plants (GUI).
Statistical analyses - To identify outperforming genes and constructs, results from the independent transformation events are evaluate for the overall influence of the gene (gene effect) and for each of the tested events (best event). Student’s t test were applied, using significance of p < 0.05 or p < 0.1. The JMP statistics software package 20 is used (Version 5.2.1, SAS Institute Inc., Cary, NC, USA).
Experimental Results
The polynucleotide sequences of the invention were assayed for a number of desired traits.
Tables 5-6 depict analyses of Leaf Area in plants overexpressing the 25 polynucleotides of the invention under the regulation of 6669 promoter under 25 % PEG conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control, with A indicating a difference at a P < 0.05 level of significance and, A* a difference at a P < 0.1 level of significance.
Table 5: Genes showing improve Leaf Area under 25 % PEG
Leaf Area [cmA2], 25% PEG
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.3 8 B 0.38 B 0.6 8 B 0.68 B
ΜΑΒΙ 0.4 9 A 0.63 A 67 0.7 2 B 6 0.80 18
MAB2 5 0.3 3 C 0.49 A 28 0.6 1 B 0.88 A 30
Table 5: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 6: Genes showing improve Leaf Area under 25% PEG
Leaf Area [cmA2], 25% PEG
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 3 B 0.23 B 0.4 4 B 0.44 B
ΜΑΒΙ 5 0.2 5 B 0.32 A 43 0.3 6 B 0.48 B 9
ΜΑΒΙ 7 0.2 7 A 0.36 A 57 0.4 6 B 0.65 A 48
ΜΑΒΙ 8 0.3 0 A 0.36 A 57 0.3 9 B 0.51 B 15
MAB3 5 0.2 1 B 0.26 B 14 0.3 8 B 0.60 A 36
Table 6: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 7-9 depict analyses of Roots Coverage in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter under 25 % PEG conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
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Table 7
Roots Coverage [cmA2], 25% PEG
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 4.3 7 B 4.37 B 6.69 B 6.69 B
ΜΑΒΙ 7.1 7 A 10.3 2 A 136 9.25 A 9.73 A 45
Table 7: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 8
Roots Coverage [cmA2], 25% PEG
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 4.0 4 B 4.04 B 11.0 9 B 11.09 B
ΜΑΒΙ 5 4.5 3 B 5.60 A 39 10.1 0 B 11.74 B 6
ΜΑΒΙ 8 5.2 3 A 6.79 A 68 9.92 B 10.29 B -7
ΜΑΒΙ 46 5.1 0 B 7.01 A 73 8.67 B 10.04 B -9
Table 8: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 9
Roots Coverage [cmA2], 25% PEG
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improve ment of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 2.1 1 B 2.11 B 5.67 B 5.67 B
MAB 18 2.0 5 B 2.75 B 30 5.40 B 8.76 A 55
MAB 32 1.9 8 B 5.06 A 140 4.31 B 10.55 A 86
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MAB 35 2.6 2 B 3.82 A 81 7.19 A* 10.04 A 77
MAB 4 3.0 3 A 5.64 A 168 7.38 A* 11.38 A 101
MAB 146 1.8 4 B 3.65 A 73 5.05 B 9.21 A 63
Table 9: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 10-11 depict analyses of Roots Length in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different 10 from the control.
Table 10
Roots Length 1cm], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 4.7 1 A 4.71 A 5.7 1 B 5.71 B
MAB 1 5.3 7 A 5.91 A 25 6.0 9 B 6.40 B 12
Table 10: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 11
Roots Length 1cm], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 2.8 8 B 2.88 B 5.1 1 B 5.11 B
MAB 18 3.2 2 B 4.29 A 49 4.8 6 B 6.33 B 24
MAB 32 2.7 4 B 5.78 A 101 3.7 5 B 7.17 A 40
MAB 35 3.3 5 A* 4.79 A 66 5.3 0 B 6.76 A 32
MAB 4 3.2 5 B 4.80 A 67 5.2 4 B 7.32 A 43
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MAB 146 2.4 3 B 4.00 A 39 4.0 4 B 6.39 A 25
Table 11: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 12-13 depict analyses of Leaf Area RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in 25 % PEG. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 12
Leaf Area RGR [cmA2/day], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 6 B 0.46 B 0.1 2 B 0.12 B
MAB 1 0.6 8 A 1.47 A 222 0.2 0 A 0.30 A 151
MAB 17 0.4 3 B 0.50 B 8 0.1 7 B 0.29 A 145
MAB 35 0.6 5 A 0.71 A 54 0.1 9 A 0.23 A 93
MAB 146 0.5 5 B 0.80 A 75 0.1 6 B 0.20 B 66
Table 12: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 13
Leaf Area RGR [cmA2/day], PEG 25%
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 9 B 0.49 B 0.2 4 B 0.24 B
MAB 6 0.8 9 A 1.60 A 226 0.2 7 B 0.33 B 39
Table 13: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
2017228711 15 Sep 2017
Tables 14-18 depict analyses of Roots Coverage RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in 25% PEG. Each Table represents an independent experiment, using 4 independent events per 5 gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 14
Roots Coverage RGR |cmA2/day|, PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 5.7 4 B 5.74 B 0.1 1 B 0.11 B
MAB 25 4.0 3 B 5.44 B -5 0.1 6 B 0.21 A 96
MAB 44 5.3 2 B 7.79 B 36 0.1 7 B 0.28 A 155
Table 14: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 15
Roots Coverage RGR |cmA2/day|, PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 3 B 0.43 B 0.3 0 B 0.30 B
MAB 1 2.1 6 A 3.09 A 621 0.3 6 B 0.43 A 44
MAB 15 1.5 5 A 2.81 A 555 0.3 0 B 0.33 B 9
MAB 17 1.9 9 A 4.08 A 852 0.3 5 B 0.53 A 78
MAB 18 1.4 4 A 1.90 A 343 0.2 9 B 0.36 B 19
MAB 35 1.1 0 B 1.71 B 298 0.3 7 B 0.48 A 59
MAB 146 2.1 6 A 4.03 A 841 0.3 0 B 0.41 A 38
Table 15: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant difference at P < 0.05, A* meaning significant difference at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 16
2017228711 15 Sep 2017
Roots Coverage RGR [cmA2/day], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 1.2 7 B 1.27 B 0.0 8 B 0.08 B
ΜΑΒΙ 00 1.2 6 B 1.52 B 19 0.1 2 B 0.19 A 131
ΜΑΒΙ 34 1.6 4 A* 2.20 A 73 0.0 8 B 0.12 B 48
ΜΑΒΙ 3 1.5 7 B 2.16 A 70 0.1 9 A 0.32 A 294
ΜΑΒΙ 5 1.6 1 A* 2.71 A 113 0.1 0 B 0.13 B 56
ΜΑΒΙ 7 2.1 5 A 2.24 A 76 0.1 3 B 0.15 B 88
ΜΑΒ3 GA 1.5 2 B 2.02 A 58 0.0 9 B 0.12 B 45
Table 16: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 17
Roots Coverage RGR |cmA2/day|, PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.9 5 B 0.95 B 0.3 0 B 0.30 B
ΜΑΒΙ 8 0.7 5 B 2.04 A 116 0.2 9 B 0.47 A 60
MAB3 5 1.4 4 A* 4.53 A 379 0.3 2 B 0.48 A 63
MAB4 1.2 8 B 2.17 A 129 0.2 9 B 0.44 A 49
ΜΑΒΙ 46 0.4 7 B 0.86 B -9 0.3 5 B 0.45 A 52
Tablel7: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 18
2017228711 15 Sep 2017
Roots Coverage RGR [cmA2/day], PEG 25%
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 1.6 6 B 1.66 B 0.2 1 B 0.21 B
MAB4 3 1.4 3 B 2.24 B 35 0.2 9 A 0.39 A 86
Table 18: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 19-21 depict analyses of Roots Length RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 19
Roots Length RGR [cm/day], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 3 B 0.23 B 0.0 9 B 0.09 B
ΜΑΒΙ 0.4 6 A 0.58 A 148 0.1 2 A 0.14 A 58
ΜΑΒΙ 5 0.4 3 A 0.58 A 148 0.0 8 B 0.10 B 16
ΜΑΒΙ 7 0.4 5 A 0.57 A 147 0.1 1 A 0.16 A 87
ΜΑΒΙ 8 0.4 1 A 0.44 A 89 0.1 0 B 0.13 A 45
ΜΑΒ3 5 0.3 1 B 0.37 A 59 0.1 0 B 0.13 A 51
ΜΑΒΙ 46 0.4 9 A 0.65 A 178 0.0 9 B 0.10 B 17
Tablel9: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 20
2017228711 15 Sep 2017
Roots Length RGR [cm/day], PEG 25%
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 0 B 0.20 B 0.0 7 B 0.07 B
ΜΑΒΙ 34 0.2 8 A 0.33 A 68 0.0 7 B 0.08 B 16
ΜΑΒΙ 3 0.3 4 A 0.46 A 133 0.1 1 A 0.15 A 113
ΜΑΒΙ 5 0.3 0 A 0.47 A 139 0.0 6 B 0.07 B 1
ΜΑΒΙ 7 0.3 9 A 0.44 A 121 0.0 9 B 0.10 B 39
ΜΑΒ3 GA 0.2 8 A 0.34 A 72 0.0 5 B 0.08 B 8
Table 20; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 21
Roots Length RGR [cm/day], PEG 25%
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 9 B 0.29 B 0.1 1 B 0.11 B
ΜΑΒΙ 37 0.2 7 B 0.39 A 32 0.1 1 B 0.12 B 11
MAB4 3 0.3 3 B 0.49 A 66 0.1 4 A 0.17 A 60
MAB5 0 0.3 7 A 0.53 A 82 0.1 3 B 0.15 A 45
MAB6 0.3 3 B 0.43 A 47 0.1 2 B 0.15 B 38
Table 21; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 22-23 depict analyses of Plant Fresh Weight in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different 15 from the control.
Table 22
2017228711 15 Sep 2017
Gene Id Plant Fresh Weight [grJ, PEG 25%
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.20 B 0.20 B
MAB15 0.25 B 0.30 A 51
MAB18 0.21 B 0.26 A 33
Table 22; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 23
Gene Id Plant Fresh Weight |gr|, PEG 25%
LSM Significance * LSM best Event Significance* % improvement of Best event
GUI 0.18 B 0.18 B
MAB 17 0.22 B 0.29 A 66
MAB3GA 0.18 B 0.27 A 53
Table 23; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 24-27 depict analyses of Leaf Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 24
Leaf Area [cmA2], Normal Conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improve ment of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 9 B 0.49 B 0.8 2 B 0.82 B
MAB 1 0.6 5 A 0.73 A 47 1.0 0 A 1.13 A 38
Table 24; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significantly different at P < 0.05. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 25
2017228711 15 Sep 2017
Leaf Area [cmA2], Normal Conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 4 B 0.24 B 0.5 6 B 0.56 B
MAB 17 0.3 1 A 0.34 A 40 0.7 3 A 0.90 A 61
MAB 18 0.2 9 A 0.37 A 52 0.6 9 A 0.79 A 42
Table 25: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 26
Leaf Area [cmA2], Normal Conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.3 9 B 0.39 B 0.9 8 B 0.98 B
MAB 15 0.4 6 A* 0.61 A 57 1.2 2 A 1.38 A 41
MAB 17 0.4 6 A* 0.57 A 47 1.1 3 A* 1.32 A 34
MAB3 G A 0.3 8 B 0.56 A 45 0.9 7 B 1.38 A 40
Table 26: LSM = Least square mean; % improvement = compare to control (GUI); ); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 27
Leaf Area [cmA2], Normal conditions
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.3 4 B 0.34 B 0.6 7 B 0.67 B
MAB 6 0.3 2 B 0.41 A 19 0.6 0 B 0.74 B 0.60
Table 27: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
2017228711 15 Sep 2017
Tables 28-31 depict analyses of Roots Coverage in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are 5 significantly different from the control.
Table 28
Roots Coverage |cmA2|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 3.3 4 B 3.34 B 11.6 1 B 11.61 B
MAB 18 3.3 1 B 4.78 A 43 10.6 6 B 13.30 B 14
Table 28; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 29
Roots Coverage |cmA2|, Normal conditions
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 5.40 B 5.40 B
MAB 100 5.05 B 7.06 A 31
Table 29: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 30
Roots Coverage |cmA2|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 3.5 3 B 3.53 B 8.52 B 8.52 B
MAB 18 4.1 7 A* 5.30 A 50 9.81 A* 12.89 A 51
MAB 32 2.5 5 B 4.71 A 33 6.40 B 12.37 A 45
2017228711 15 Sep 2017
MAB 35 3.7 3 B 4.59 A 30 8.55 B 11.12 A 30
MAB 46 2.4 6 B 3.42 B -3 6.55 B 10.98 A 29
MAB 146 2.3 3 B 3.95 B 12 7.05 B 10.86 A 28
Table 30: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 31
Roots Coverage |cmA2|, Normal conditions
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LSM best Even t Signif! cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 3.7 3 B 3.73 B 7.11 B 7.11 B
MAB 6 3.6 3 B 4.94 A 33 6.30 B 8.00 B 13
Table 31: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 32-33 depict analyses of Roots Length in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 32
Roots Length |cm|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 5.8 9 B 5.89 B 6.8 2 B 6.82 B
ΜΑΒΙ 6.7 3 A 7.39 A 26 7.0 2 B 7.63 B 12
MAB 10 5.4 5 B 8.07 A 37 5.8 3 B 8.18 B 20
2017228711 15 Sep 2017
Table 32: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 33
Roots Length (cm], Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 3.9 6 B 3.96 B 6.5 1 B 6.51 B
MAB 18 5.0 7 A 5.70 A 44 7.0 8 A 8.03 A 23
MAB 32 3.6 8 B 6.12 A 55 5.8 2 B 8.22 A 26
MAB 35 4.5 8 A 5.76 A 46 6.7 7 B 7.75 A 19
MAB 46 3.3 9 B 4.31 B 9 5.5 5 B 7.42 A 14
MAB 146 3.1 4 B 4.82 A 22 5.4 7 B 7.48 A 15
Table 33: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 34-36 depict analyses of Leaf Area RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 34
Leaf Area RGR [cm/day], Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 3 B 0.43 B 0.2 0 B 0.20 B
MAB 15 0.7 9 A 1.25 A 189 0.2 1 B 0.27 B 36
MAB 146 0.6 2 B 0.97 A 124 0.1 5 C 0.18 B -13
2017228711 15 Sep 2017
Table 34: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 35
Leaf Area RGR |cm/day], Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.7 3 B 0.73 B 0.2 1 B 0.21 B
ΜΑΒΙ 00 0.7 2 B 1.00 A 37 0.2 7 B 0.32 A 48
ΜΑΒΙ 34 0.8 5 B 0.92 B 27 0.3 1 A 0.37 A 75
ΜΑΒΙ 5 0.8 8 A* 1.24 A 70 0.2 8 B 0.33 A 56
ΜΑΒΙ 7 0.9 1 A 1.18 A 62 0.2 6 B 0.33 A 55
ΜΑΒ3 GA 0.8 8 B 1.16 A 59 0.2 7 B 0.31 B 46
Table 35: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P <0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 36
Leaf Area RGR [cm/day], Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.9 2 B 0.29 B 0.29 B
MAB 32 0.9 5 B 1.31 A 43 0.28 B 0.31 B 5
Table 36: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 37-41 depict analyses of Roots Coverage RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 37
Roots Coverage RGR [cm/day], Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 5.6 2 B 5.62 B 0.1 8 B 0.18 B
MAB 10 7.6 9 B 15.1 0 A 168 0.0 8 B 0.14 B -20
MAB 44 5.2 8 B 11.6 9 A 108 0.1 3 B 0.17 B -5
Table 37: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 38
Roots Coverage RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 3 B 0.23 B 0.4 0 B 0.40 B
MAB 1 0.9 0 A 1.23 A 444 0.3 3 B 0.42 B 7
MAB 15 1.0 6 A 1.65 A 628 0.3 4 B 0.42 B 6
MAB 18 0.9 4 A 1.76 A 677 0.3 7 B 0.52 B 32
MAB 35 0.5 6 B 1.00 A 342 0.3 8 B 0.41 B 3
MAB 146 0.8 0 A 1.09 A 381 0.3 5 B 0.50 B 26
Table 38; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 39
Roots Coverage RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 1.6 4 B 1.64 B 0.1 2 B 0.12 B
2017228711 15 Sep 2017
MAB 134 3.0 9 A 4.38 A 167 0.1 4 B 0.17 B 35
MAB 13 2.4 7 A 2.82 A 72 0.1 1 B 0.13 B 6
MAB 15 1.9 6 B 2.75 A 68 0.1 5 B 0.16 B 33
MAB 17 2.0 9 B 3.09 A 89 0.1 5 B 0.20 A 60
Table 39: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 40
Roots Coverage RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 2.5 3 B 2.53 B 0.2 4 B 0.24 B
MAB 35 1.6 6 B 4.14 A 63 0.2 9 B 0.54 A 123
MAB 4 1.4 6 B 2.64 B 4 0.3 2 B 0.42 A 73
MAB 146 0.6 2 B 0.95 B -63 0.4 1 A 0.75 A 207
Table 40: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 41
Roots Coverage RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 1.0 8 B 1.08 B 0.3 1 B 0.31 B
MAB 137 1.3 6 B 2.03 A 88 0.2 6 B 0.31 B 1
MAB 43 1.3 9 B 2.35 A 118 0.2 3 B 0.27 B -12
MAB 50 1.5 7 A 1.98 A 83 0.2 7 B 0.30 B -3
MAB 6 1.1 6 B 1.94 A 80 0.2 5 B 0.29 B -6
MAB 99 1.4 8 A 2.63 A 144 0.2 1 B 0.27 B -13
2017228711 15 Sep 2017
Table 41: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 42-46 depict analyses of Roots Length RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 42
Roots Length RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 1.07 B 1.07 B
MAB 10 1.29 B 2.01 A 88
Table 42: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 43
Roots Length RGR [cm/day], Normal conditions
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.17 B 0.17 B
ΜΑΒΙ 0.26 A 0.34 A 93
MAB15 0.32 A 0.45 A 156
MAB17 0.24 A 0.28 A 61
MAB18 0.30 A 0.41 A 136
MAB 146 0.26 A 0.34 A 93
Table 43: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 44
Roots Length RGR |cm/day|, Normal conditions
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 9 B 0.29 B 0.0 8 B 0.08 B
MAB 100 0.3 6 B 0.39 B 31 0.0 8 B 0.13 A 67
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MAB 134 0.5 1 A 0.63 A 115 0.0 8 B 0.09 B 23
MAB 13 0.5 0 A 0.61 A 107 0.0 8 B 0.09 B 19
MAB 15 0.4 0 A 0.53 A 79 0.0 8 B 0.09 B 19
MAB 17 0.3 8 A* 0.44 A 49 0.1 0 A 0.13 A 70
Table 44: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 45
Roots Length RGR |cm/day|, Normal conditions
Gene Id Day 14 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.11 B 0.11 B
MAB32 0.11 B 0.15 A 35
MAB35 0.11 B 0.20 A 76
MAB4 0.11 B 0.17 A 50
MAB 146 0.15 A 0.19 A 71
Table 45: LSM = Least square mean; % improvement = compare to control (GUI); ); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 46
Roots Length RGR [cm/day], Normal conditions
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.3 1 B 0.31 B 0.1 2 B 0.12 B
MAB 137 0.3 3 B 0.40 A 31 0.1 1 B 0.12 B -1
MAB 43 0.3 3 B 0.44 A 41 0.1 1 B 0.12 B -2
MAB 50 0.3 9 A 0.42 A 35 0.1 3 B 0.17 A 34
MAB 6 0.3 0 B 0.41 A 33 0.1 2 B 0.18 A 41
Table 46: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 47-48 depict analyses of Plant Fresh Weight in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in normal conditions. Each Table represents an independent experiment, using 4 independent
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100 events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 47
Plant Fresh Weight |gr|, Normal conditions
Gene Id Day 14 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.15 B 0.15 B
MAB 15 0.24 A 0.28 A 93
MAB 17 0.21 A 0.25 A 73
MAB 18 0.22 A 0.29 A 101
Table 47: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 48
Plant Fresh Weight [gr], Normal conditions
Gene Id Day 14 from planting
LSM Significance * LSM best Event Significance* % improvement of Best event
GUI 0.20 B 0.20 B
MAB 100 0.28 A* 0.33 A 62
MAB 134 0.23 B 0.34 A 64
MAB 13 0.31 A 0.35 A 73
MAB 15 0.38 A 0.42 A 106
MAB 17 0.37 A 0.53 A 159
MAB3GA 0.28 A* 0.40 A 94
Table 48: LSM = Least square mean; % improvement = compare to control (GUI); ); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Assay 2: plant growth at Nitrogen deficiency under Tissue culture conditions The present inventors have found the NUE (Nitrogen Utilization Efficiency) assay to be relevant for the evaluation of the ABST candidate genes, since NUE deficiency encourages root elongation, increase of root coverage and allows detecting the potential of the plant to generate a better root system under drought conditions. In addition, there are indications in the literature (Wesley et al., 2002 Journal of Experiment Botany Vol. 53, No. 366, pp. 13-25) that biological mechanisms of NUE and drought tolerance are linked.
Surface sterilized seeds are sown in basal media [50 % Murashige-Skoog medium (MS) supplemented with 0.8 % plant agar as solidifying agent] in the presence of Kanamycin (for selecting only transgenic plants). After sowing, plates are
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101 transferred for 2-3 days at 4 °C for stratification and then grown at 25 °C under 12-hour light 12-hour dark daily cycles for 7 to 10 days. At this time point, seedlings randomly chosen are carefully transferred to plates holding nitrogen-limiting conditions: 0.5 MS media in which the combined nitrogen concentration (NH4NO3 and KNO3) is 0.75 mM (nitrogen deficient conditions) or to plates holding normal nitrogen conditions: 0.5 MS media in which the combined nitrogen concentration (NH4NO3 and KNO3) is 3 mM (normal nitrogen concentration). All tissue culture experiments were grown at the same time (NUE, PEG and Normal). Results for growth under normal conditions for NUE are the same as for PEG and are presented in assay 1. Each plate contains 5 seedlings of the same event, and 3-4 different plates (replicates) for each event. For each polynucleotide of the invention at least four independent transformation events are analyzed from each construct. Plants expressing the polynucleotides of the invention are compared to the average measurement of the control plants (GUI- harboring the GUS gene under the same promoter) used in the same experiment.
Digital imaging and statistical analysis - Parameters were measured and analyzed as described in Assay 1 above.
Experimental Results - The polynucleotide sequences of the invention were assayed for a number of desired traits.
Tables 49-53 depict analyses of Leaf Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B) are significantly different from the control.
Table 49
Leaf Area [cmA2], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 5 B 0.45 B 0.4 1 B 0.41 B
ΜΑΒΙ 0.4 9 B 0.65 A 44 0.5 0 A 0.55 A 35
ΜΑΒΙ 0 0.4 6 B 0.62 A 38 0.5 1 A 0.69 A 68
MAB6 0.4 2 B 0.53 B 17 0.4 9 B 0.61 A 49
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102
Table 49: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P <0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 50
Leaf Area 1«ηΛ2], NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 3 B 0.23 B 0.4 1 B 0.41 B
MAB 1 0.2 2 B 0.24 B 5 0.5 0 A 0.55 A 35
MAB 15 0.2 5 B 0.32 A 43 0.5 1 A 0.69 A 68
MAB 17 0.2 7 A 0.36 A 57 0.5 5 A 0.70 A 72
MAB 18 0.3 0 A 0.36 A 57 0.5 9 A 0.73 A 80
MAB 35 0.2 1 B 0.26 B 14 0.4 9 B 0.61 A 49
MAB 146 0.2 6 B 0.28 B 23 0.5 5 A 0.60 A 48
Table 50: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 51
Leaf Area 1«ηΛ2], NEE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.34 B 0.34 B
MAB 17 0.32 B 0.44 A 31
MAB3GA 0.32 B 0.44 A 31
Table 51: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 52
Leaf Area 1«ηΛ2], NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 1 B 0.21 B 0.6 3 B 0.63 B
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MAB 18 0.2 3 B 0.31 A 50 0.5 8 B 0.77 A 22
MAB 4 0.2 0 B 0.31 A 48 0.5 4 B 0.82 A 30
MAB 146 0.2 1 B 0.29 A 41 0.4 8 C 0.59 B -6
Table 52: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 53
Leaf Area (cmA2], NUE 0.75 mM
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 7 B 0.27 B 0.5 1 B 0.51 B
MAB4 3 0.2 5 B 0.35 A 29 0.4 7 B 0.60 B 18
MAB5 0 0.2 8 B 0.32 B 19 0.5 4 B 0.66 A 31
MAB6 0.2 8 B 0.35 A 28 0.5 4 B 0.69 A 35
MAB6 6 0.2 8 B 0.34 A 25 0.5 1 B 0.59 B 17
MAB9 9 0.2 7 B 0.35 A 28 0.5 1 B 0.59 B 16
Table 53: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 54-57 depict analyses of Roots Coverage in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) 15 are significantly different from the control.
Table 54
Roots Coverage |cmA2|, NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 6.1 8 B 6.18 B 14.3 6 B 14.36 B
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104
ΜΑΒΙ 7.3 3 B 8.56 A 39 13.1 8 B 16.22 B 13
ΜΑΒΙ 0 7.9 3 A 10.3 8 A 68 13.3 2 B 14.67 B 2
MAB2 5 5.8 3 B 6.93 B 12 11.1 2 A 13.90 B -3
MAB4 4 5.3 7 B 9.93 A 61 11.1 4 A 17.59 B 22
MAB6 6.8 8 B 9.31 A 51 12.7 9 B 15.66 B 9
Table 54: LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 55
Roots Coverage |cmA2|, NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 4.0 4 B 4.04 B 12.2 4 B 12.24 B
MAB 15 4.5 3 B 5.60 A 39 13.7 0 B 16.40 A 34
MAB 17 4.1 5 B 4.85 B 20 13.1 6 B 15.06 A 23
MAB 18 5.2 3 A 6.79 A 68 14.4 7 A 15.52 A 27
MAB 35 4.0 3 B 4.90 B 21 13.9 5 B 15.62 A 28
MAB 146 5.1 0 B 7.01 A 73 14.6 5 A 15.70 A 28
Table 55; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 56
Roots Coverage [cmA2], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 3.1 4 B 3.14 B 10.8 8 B 10.88 B
MAB 18 5.3 9 A 7.64 A 144 12.7 6 B 16.64 A 53
MAB 32 3.5 8 B 7.13 A 127 9.79 B 16.22 A 49
MAB 35 5.0 0 A 6.49 A 107 13.3 1 A 15.36 A 41
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105
MAB 4 4.1 6 B 7.34 A 134 12.0 0 B 16.52 A 52
MAB 46 3.0 1 B 3.78 B 21 8.35 C 12.09 B 11
MAB 146 4.2 2 B 7.34 A 134 11.4 8 B 14.98 A 38
Table 56; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 57
Roots Coverage |cmA2|, NUE 0.75 mM
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LSM Signi fican ce* LSM best Event Significa nee* % improve ment of Best event
GUI 4.5 6 B 4.56 B 9.81 B 9.81 B
MAB6 5.6 6 A 7.98 A 75 10.6 1 B 14.87 A 52
MAB6 6 5.8 3 A 6.58 A 44 10.3 1 B 11.49 B 17
Table 57; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 58-61 depict analyses of Roots Length in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 58
Roots I .ength 1cm], NEE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 6.31 B 6.31 B
MAB44 5.34 B 7.07 A 12
Table 58; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
106
Table 59
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Roots Length [cm], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 4.5 5 B 4.55 B 7.2 3 B 7.23 B
MAB 15 4.4 8 B 5.40 A 19 6.9 3 B 7.49 B 4
MAB 18 4.6 1 B 5.48 A 20 7.5 9 B 7.86 B 9
MAB 146 4.7 0 B 5.20 B 14 7.6 6 B 7.95 A 10
Table 59; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 60
Roots Length |cm|, NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 3.6 1 B 3.61 B 6.1 5 B 6.15 B
ΜΑΒΙ 8 4.9 3 A 6.44 A 79 7.3 0 A 8.11 A 32
MAB3 2 4.0 2 B 6.48 A 80 6.5 3 B 8.51 A 38
MAB3 5 4.7 0 A 5.47 A 52 7.2 0 A 7.46 A 21
MAB4 4.0 6 A* 5.54 A 54 6.6 0 B 8.02 A 30
ΜΑΒΙ 46 3.7 7 B 5.54 A 54 6.0 9 B 7.19 A 17
Table 60; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 61
Roots Length |cm|, NEE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 4.87 B 4.87 B
MAB66 5.27 B 5.74 A 18
Table 61; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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107
Tables 62-64 depict analyses of Leaf Area RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) 5 are significantly different from the control.
Table 62
Leaf area RGR |cm/day|, NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 6 B 0.46 B 0.1 2 B 0.12 B
MAB 1 0.6 8 A 1.47 A 222 0.2 0 A 0.30 A 151
MAB 17 0.4 3 B 0.50 B 8 0.1 7 B 0.29 A 145
MAB 35 0.6 5 A 0.71 A 54 0.1 9 A 0.23 A 93
MAB 146 0.5 5 B 0.80 A 75 0.1 6 B 0.20 B 66
Table 62; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 63
Leaf area RGR [cm/day], NEE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.80 B 0.80 B
MAB 18 0.87 B 1.24 A 56
MAB32 0.94 B 1.53 A 91
MAB35 0.96 B 1.21 A 51
MAB4 0.71 B 0.81 B 1
MAB46 0.64 B 0.75 B -7
MAB 146 0.82 B 1.04 B 30
Table 63; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
108
Table 64
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Leaf area RGR [cm/day], NUE 0.75 mM
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 1.2 2 B 1.22 B 0.2 8 B 0.28 B
ΜΑΒΙ 37 2.1 2 B 5.12 A 319 0.2 9 B 0.35 B 25
MAB4 3 1.9 4 B 5.18 A 323 0.2 9 B 0.35 B 28
MAB5 0 1.1 5 B 1.76 B 44 0.3 2 B 0.41 A 50
Table 64; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 65-69 depict analyses of Roots Coverage RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B, 10 C) are significantly different from the control.
Table 65
Roots Coverage RGR [cm/day], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 5.3 5 B 5.35 B 0.2 8 B 0.28 B
MAB 25 7.3 8 B 11.6 2 A 117 0.1 9 C 0.26 B -6
MAB 44 7.1 9 B 11.5 2 A 115 0.2 6 B 0.35 B 23
Table 65; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
109
Table 66
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Roots Coverage RGR [cm/day], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.4 3 B 0.43 B 0.3 0 B 0.30 B
MAB 1 2.1 6 A 3.09 A 621 0.3 6 B 0.43 A 44
MAB 15 1.5 5 A 2.81 A 555 0.3 0 B 0.33 B 9
MAB 17 1.9 9 A 4.08 A 852 0.3 5 B 0.53 A 78
MAB 18 1.4 4 A 1.90 A 343 0.2 9 B 0.36 B 19
MAB 35 1.1 0 B 1.71 B 298 0.3 7 B 0.48 A 59
MAB 146 2.1 6 A 4.03 A 841 0.3 0 B 0.41 A 38
Table 66; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 67
Roots Coverage RGR |cm/day|, NUE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 2.30 B 2.30 B
MAB 100 2.85 B 4.02 A 74
MAB 134 4.27 A 5.99 A 160
MAB13 3.95 A 4.84 A 110
MAB15 3.05 A* 3.97 A 73
MAB17 2.96 B 3.76 A 63
Table 67; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 68
Roots Coverage RGR [cm/day], NUE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 2.2 8 B 2.28 B 0.4 4 B 0.44 B
MAB3 5 2.0 2 B 4.82 A 111 0.3 3 B 0.53 B 20
MAB4 1.8 0 B 2.90 B 27 0.4 0 B 0.63 A 42
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110
Table 68; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 69
Roots Coverage RGR [cm/day], NUE 0.75 mM
Gene Id Day 7 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 1.60 B 1.60 B
MAB137 2.19 A 2.55 B 60
MAB43 2.00 B 2.75 A 72
MAB50 2.26 A 3.28 A 105
MAB6 2.45 A 2.96 A 85
MAB66 1.81 B 2.87 A 80
MAB99 2.25 A 3.73 A 133
Table 69; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 70-74 depict analyses of Roots Length RGR in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 70
Roots Length RGR |cm/day|, NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.9 9 B 0.99 B 0.0 4 B 0.04 B
MAB 44 1.1 0 B 1.64 A 65 0.0 6 B 0.09 A 108
Table 70; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 71
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Roots Length RGR [cm/day], NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.2 3 B 0.23 B 0.0 9 B 0.09 B
MAB 1 0.4 6 A 0.58 A 148 0.1 2 A 0.14 A 58
MAB 15 0.4 3 A 0.58 A 148 0.0 8 B 0.10 B 16
MAB 17 0.4 5 A 0.57 A 147 0.1 1 A 0.16 A 87
MAB 18 0.4 1 A 0.44 A 89 0.1 0 B 0.13 A 45
MAB 35 0.3 1 B 0.37 A 59 0.1 0 B 0.13 A 51
Table 71; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 72
Roots Length RGR |cm/day|, NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LSM Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.35 B 0.35 B 0.0 6 B 0.06 B
MA B100 0.46 A 0.61 A 73 0.0 8 B 0.11 A 80
MA B134 0.62 A 0.73 A 107 0.0 9 A 0.10 A 60
MA B13 0.69 A 0.84 A 140 0.0 8 B 0.11 A 66
MA B15 0.52 A 0.58 A 66 0.0 7 B 0.09 B 44
MA B17 0.52 A 0.64 A 81 0.0 8 B 0.09 A 44
MA B3 GA 0.44 B 0.51 A 46 0.0 7 B 0.09 B 38
Table 72; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 73
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Roots Length RGR [cm/day], NEE 0.75 mM
Gene Id Day 7 from planting Day 14 from planting
LS M Signific ance* LSM best Even t Signifi cance * % improv ement of Best event LS M Signifi cance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.6 1 B 0.61 B 0.1 2 B 0.12 B
MAB 35 0.5 2 B 0.91 A 48 0.1 0 B 0.16 B 29
MAB 4 0.5 3 B 0.65 B 6 0.1 2 B 0.19 A 52
MAB 146 0.3 7 C 0.42 B -31 0.1 2 B 0.17 A 39
Table 73; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 74
Roots Length RGR |cm/day|, NEE 0.75 mM
Gene Id Day 7 from planting Day 10 from planting
LS M Signific ance* LS M best Eve nt Signif icance * % improv ement of Best event LS M Signif icance * LSM best Event Significa nee* % improve ment of Best event
GUI 0.3 6 B 0.36 B 0.1 1 B 0.11 B
ΜΑΒΙ 37 0.4 6 A 0.55 A 52 0.1 3 B 0.18 A 72
MAB4 3 0.4 1 B 0.53 A 47 0.1 2 B 0.14 B 30
MAB5 0 0.4 8 A 0.57 A 59 0.1 2 B 0.16 A 46
MAB6 0.5 3 A 0.64 A 79 0.1 0 B 0.12 B 9
MAB6 6 0.4 1 B 0.55 A 54 0.1 0 B 0.12 B 9
MAB9 9 0.4 7 A 0.62 A 74 0.1 0 B 0.13 B 19
Table 74; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 75-76 depict analyses of Plant Fresh Weight in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter in nitrogen deficient conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) 15 are significantly different from the control.
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Table 75
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Plar it Fresh Weight [gr], NUE 0.75 mM
Gene Id Day 14 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.15 B
ΜΑΒΙ 0.25 A 0.46 A 208
MAB6 0.20 B 0.29 A 95
Table 75; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 76
Plar it Fresh Weight |gr|, NEE 0.75 mM
Gene Id Day 10 from planting
LSM Significance* LSM best Event Significance* % improvement of Best event
GUI 0.15 B
MAB137 0.18 A 0.19 A 31
MAB50 0.16 B 0.22 A 49
MAB6 0.16 B 0.22 A 52
MAB66 0.15 B 0.19 A 32
Table 76; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
EXAMPLE 7
IMPRO VED ABSTIN GREENHOUSE ASSA Y
ABS tolerance: Yield and plant growth rate at high salinity concentration under greenhouse conditions - This assay follows the rosette area growth of plants grown in the greenhouse as well as seed yield at high salinity irrigation. Seeds were sown in agar media supplemented only with a selection agent (Kanamycin) and Hoagland solution under nursery conditions. The T2 transgenic seedlings are then transplanted to 1.7 trays filled with peat and perlite. The trails were irrigated with tap water (provided from the pots’ bottom). Half of the plants are irrigated with a salt solution (40-80 mM NaCl and 5 mM CaCl2) to induce salinity stress (stress conditions). The other half of the plants are continued to be irrigated with tap water (normal conditions). All plants are grown in the greenhouse until plants reach the mature seeds stage, then harvested (the above ground tissue) and weighted (immediately or following drying in oven at 50 °C for 24 hour). High salinity conditions are achieved by irrigation with a solution containing 40-80 mM NaCl (ABS growth conditions) and are compared to regular growth conditions.
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The plants were analyzed for their overall size, growth rate, seed yield, and weight of 1,000 seeds, dry matter and harvest index (HI- seed yield / dry matter). Transgenic plants performance was compared to control plants grown in parallel under the same conditions. Mock- transgenic plants expressing the uidA reporter gene (GUS Intron - GUI) under the same promoter were used as control.
The experiment is planned in nested randomized plot distribution. High salinity conditions are achieved by irrigation with a solution containing 40-80 mM NaCl (ABS growth conditions).
Digital imaging - A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which included 4 light units (4x150 Watts light bulb) was used for capturing images of plantlets.
The image capturing process was repeated every 2-3 days starting at day 1 after sowing till day 10. The same camera attached with a 24 mm focal length lens (Canon EF series), placed in a custom made iron mount, was used for capturing images of larger plants sawn in white tubs in an environmental controlled greenhouse (as seen on Figures 2a-b). The tubs were square shape include E7 liter trays. During the capture process, the trays were placed beneath the iron mount, while avoiding direct sun light and casting of shadows. This process was repeated every 2-3 days for up to 10 days.
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ E37 (Java based image processing program which was developed at the U.S National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/). Images were captured in resolution of 6 Mega Pixels (3072 x 2048 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Vegetative parameters analysis - Using the digital analysis leaves data was calculated, including leaf Average area, Rosette diameter and rosette area. The Relative Growth Rate (RGR) for the rosette parameters was calculated according to Formula I as described in Example 6. On day 80 from sowing, the plants were harvested and left to dry at 30 °C in a drying chamber. The biomass and seed weight of each plot was separated, measured and divided by the number of plants. Dry weight = total weight of
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115 the vegetative portion above ground (excluding roots) after drying at 30 °C in a drying chamber; Seed yield per plant = total seed weight per plant (gr).
The weight of 1000 seeds was determine as follows: seeds were scattered on a glass tray and a picture was taken. Each sample was weighted and then using the digital analysis, the number of seeds in each sample was calculated. 1000 seeds weight was calculated using formula II:
Formula II
1000 Seed Weight = number of seed in sample/ sample weight X 1000
Harvest Index - The harvest index was calculated using Formula III
Formula III:
Harvest Index = Average seed yield per plant/ Average dry weight
Each construct is validated in its T2 generation. Transgenic plants expressing the uidA reporter gene (GUI) under the same promoter are used as control.
Statistical analyses - To identify genes conferring significantly improved tolerance to abiotic stresses or enlarged root architecture, the results obtained from the transgenic plants are compared to those obtained from control plants. To identify outperforming genes and constructs, results from the independent transformation events tested are analyzed separately. In addition, genes and constructs are also analyzed taking into consideration the results obtained from all the independent transformation events tested the specific construct. For gene versus control analysis Student’s t test were applied, using significance of P < 0.05 or P < 0.1. The JMP statistics software package is used (Version 5.2.1, SAS Institute Inc., Cary, NC, USA).
Experimental Results
The polynucleotide sequences of the invention were assayed for a number of desired traits.
Tables 77-86 depict analyses of Rosette Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
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Table 77
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Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.58 B 0.58 B
MAB20 0.59 B 0.84 A 43
MAB50 0.57 B 0.88 A 51
Table 77; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 78
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significanc e % improvement of best event
GUI 1.27 B 1.27 B
MAB20 1.20 B 1.73 a 36
MAB50 1.21 B 2.04 a 61
Table 78; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 79
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 3.62 B 3.62 B
MAB20 3.97 B 5.18 A 43
MAB50 3.88 B 6.11 A 69
Table 79; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 80
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 7.22 B 7.22 B
MAB50 6.75 B 10.18 A 41
Table 80; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 81
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Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significanc e % improvement of best event
GUI 1.63 B 1.63 B
ΜΑΒΙ 2.03 A 2.29 A 40
MAB6 1.34 B 2.40 A 47
Table 78; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 82
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.88 B 2.88 B
ΜΑΒΙ 3.41 A* 3.76 A 31
Table 82; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1; . The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 83
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.73 B 0.73 B
ΜΑΒΙ 0.77 B 0.91 A 25
Table 83; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 84
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significanc e % improvement of best event
GUI 1.41 B 1.41 B
ΜΑΒΙ 1.62 A* 2.02 A 44
MAB 17 1.14 B 1.80 A 28
Table 84; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 85
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Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.37 B 2.37 B
ΜΑΒΙ 2.59 B 3.56 A 50
MAB 13 2.45 B 3.44 A 45
MAB 17 1.96 C 3.10 A 31
Table 85; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 86
Gene Id Rosette Area [cmA2] 80 mM NaCl, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.67 B 4.67 B
ΜΑΒΙ 5.37 A* 7.93 A 70
MAB 15 4.78 B 6.08 A 30
MAB 17 4.02 B 6.19 A 32
MAB3GA 4.39 B 6.07 A 30
Table 86; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 87-96 depict analyses of Rosette Diameter in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 87
Gene Id Rosette Diameter [cm] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.50 B 1.50 B
MAB50 1.35 B 1.80 A 20
Table 87; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 88
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Gene Id Rosette Diameter [cm] 80 mM NaCl Day 5 from planting
LSM Significance LSM of Best event Significanc e % improvement of best event
GUI 2.05 B 2.05 B
MAB50 1.82 C 2.44 A 19
Table 88; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 89
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 3.23 B 3.23 B
MAB50 3.16 B 4.12 A 27
Table 89; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 90
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.47 B 4.47 B
MAB50 4.20 B 5.31 A 19
Table 90; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 91
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.25 B 2.25 B
ΜΑΒΙ 2.60 A 2.78 A 23
Table 91; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 92
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Gene Id Rosette Diameter [cm] 80 mM NaCl Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.87 B 2.87 B
ΜΑΒΙ 3.27 A* 9.25 A 223
MAB20 2.63 B 9.69 A 238
MAB6 2.51 B 10.00 A 249
Table 92; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 93
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.90 B 4.90 B
MAB6 4.35 B 6.26 A 28
Table 93; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 94
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.05 B 2.05 B
ΜΑΒΙ 2.22 B 2.55 A 25
Table 94; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 95
Gene Id Rosette Diameter [cm] 80 mM NaCl Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.56 B 2.56 B
ΜΑΒΙ 2.78 B 3.29 A 29
MAB3GA 2.56 B 3.04 A 19
Table 95; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 96
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Gene Id Rosette Diameter [cm] 80 mM NaCl Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 3.52 B 3.52 B
ΜΑΒΙ 3.79 B 4.76 A 35
MAB 17 3.24 B 4.14 A 17
MAB3GA 3.44 B 4.12 A 17
Table 96; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 97-105 depict analyses of Leaf Average Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 97
Gene Id Leaf Average Area [cmA2] 80 mM NaCl Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.10 B 0.10 B
MAB25 0.10 B 0.13 A 30
Table 97; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 98
Gene Id Leaf Average Area [cmA2] 80 mM NaCl Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.16 B 0.16 B
MAB50 0.15 B 0.23 A 45
Table 98; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 99
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Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.45 B 0.45 B
MAB50 0.41 B 0.61 A 34
Table 99; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 100
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.74 B 0.74 B
MAB50 0.66 B 0.92 A 25
Table 100; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at p < 0.05, A* meaning significant different at p < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 101
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.20 B 0.20 B
ΜΑΒΙ 0.25 A 0.28 A 43
MAB6 0.18 B 0.30 A 51
MAB7 0.23 B 0.27 A 36
Table 101; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 102
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.69 B 0.69 B
ΜΑΒΙ 0.80 A* 0.86 A* 24
MAB20 0.62 B 0.87 A 25
MAB6 0.59 B 0.99 A 44
Table 102; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
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Table 103
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.20 B 0.20 B
ΜΑΒΙ 0.22 B 0.27 A 30
MAB17 - - 0.25 A 21
Table 103; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 104
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.28 B 0.28 B
ΜΑΒΙ 0.30 B 0.37 A 33
MAB 17 0.24 B 0.34 A 22
Table 104; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 105
Gene Id Leaf Average Area [cmA2] 80 mM NaCl, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.49 B 0.49 B
ΜΑΒΙ 0.55 B 0.76 A 53
MAB15 0.52 B 0.63 A 26
MAB17 0.45 B 0.64 A 28
Table 105; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 106-111 depict analyses of RGR Rosette Area [cmA2] of plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
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Table 106
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Gene Id RGR of Rosette Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.73 B 0.73 B
MAB 10 1.21 B 1.86 A 156
MAB 14 1.31 B 1.80 A 149
MAB2 1.59 A 2.24 A 208
MAB20 1.87 A 2.33 A 221
MAB25 1.44 A 1.63 A* 125
MAB36 1.49 A 1.89 A 161
MAB43 1.73 A 3.85 A 430
MAB44 1.76 A 2.51 A 246
MAB50 1.37 A* 1.57 A* 117
MAB9 1.47 A 1.75 A 141
Table 106; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 107
Gene Id RGR of Rosette Area [cmA2] 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.61 B 0.61 B
MAB 10 0.75 A* 0.91 A 50
MAB 14 0.79 A 0.86 B 42
MAB19 0.78 A 0.85 A 41
MAB2 0.80 A 0.93 A 54
MAB20 0.79 A 0.98 A 61
MAB36 0.83 A 0.95 A 56
MAB44 0.75 A* 0.84 A 38
MAB50 0.76 A* 0.83 B 38
MAB6 0.82 A 0.99 A 64
MAB7 0.78 A 0.87 A 44
MAB9 0.77 A 0.84 A 38
Table 107; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 108
Gene Id RGR of Rosette Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.38 B 0.38 B
MAB6 0.37 B 0.51 A 33
Table 108; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
125
Table 109
2017228711 15 Sep 2017
Gene Id RGR of Rosette Area [cmA2]
80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.88 B 0.88 B
MAB 18 0.99 A* 1.24 A 41
Table 109; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 110
Gene Id RGR of Rosette Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.47 B 0.47 B
ΜΑΒΙ 0.55 A 0.64 A 38
MAB 13 0.52 A 0.54 A* 16
MAB 17 0.52 A 0.54 A* 17
MAB 18 0.53 A 0.58 A 24
MAB3 GA 0.53 A 0.62 A 33
MAB32 0.52 A* 0.54 A* 17
MAB35 0.54 A 0.57 A 22
MAB4 0.51 A* 0.51 A* 10
MAB46 0.52 A* 0.55 A 19
MAB 146 0.54 A 0.55 A 19
MAB99 0.53 A 0.57 A 23
Table 110; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 111
Gene Id RGR of Rosette Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.49 B 0.49 B
ΜΑΒΙ 0.53 B 0.62 A 27
MAB35 0.57 A* 0.59 A* 22
MAB46 0.55 B 0.63 A 30
Table 111; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 112-118 depict analyses of RGR of Rosette Diameter in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
126
Table 112
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.28 B
MAB2 0.41 B 0.80 A 184
MAB43 0.46 B 0.83 A 195
MAB44 0.40 B 0.73 A 160
Table 112; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 113
Gene Id RGR of Rosette Diameter [cm])
80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.19 B 0.19 B
ΜΑΒΙ 0.22 B 0.24 B 25
MAB 10 0.25 A 0.29 A 49
MAB 14 0.23 A 0.25 A 31
MAB 19 0.24 A 0.26 A 37
MAB2 0.24 A 0.26 A 34
MAB20 0.25 A 0.29 A 52
MAB25 0.24 A 0.27 A 42
MAB36 0.25 A 0.28 A 45
MAB43 0.22 B 0.25 B 28
MAB50 0.25 A 0.28 A 46
MAB6 0.24 A 0.27 A 41
MAB7 0.22 B 0.27 A 38
MAB9 0.23 A 0.26 A 34
Table 113; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 114
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.14 B 0.14 B
MAB 10 0.14 B 0.31 A 122
MAB20 0.13 B 0.21 A 49
MAB25 0.15 B 0.33 A 138
MAB9 0.15 B 0.20 A 45
Table 114; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
2017228711 15 Sep 2017
127
Table 115
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.21 B 0.21 B
MAB20 0.23 B 0.34 A 67
MAB9 0.22 B 0.44 A 114
Table 115; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 116
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.34 B 0.34 B
MAB 18 0.37 B 0.46 A 35
MAB3 GA 0.34 B 0.43 A 26
MAB35 0.43 A 0.55 A 62
MAB46 0.39 B 0.49 A 42
MAB99 0.34 B 0.43 A 26
Table 116; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 117
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.16 B 0.16 B
ΜΑΒΙ 0.22 A 0.26 A 66
MAB 18 0.20 A* 0.23 A* 44
MAB46 0.25 A 0.45 A 185
MAB 146 0.20 A* 0.22 A* 42
Table 117; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 118
Gene Id RGR of Rosette Diameter [cm]) 80 mM NaCl, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.08 B 0.08 B
MAB35 0.10 B 0.13 A 57
MAB46 0.10 B 0.14 A 64
MAB 146 0.10 B 0.14 A 66
MAB99 0.10 B 0.13 A 56
Table 118; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the
2017228711 15 Sep 2017
128 cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Tables 119-121 depict analyses of RGR of Leaf Average Area [cmA2] in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 119
Gene Id RGR of Mean(Leaf Average Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.35 B 0.35 B
MAB 14 0.34 B 0.63 A 82
MAB25 0.44 B 0.83 A 137
MAB36 0.43 B 0.77 A 120
MAB6 0.24 B 0.70 A 102
Table 119; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 120
Gene Id RGR of Mean(Leaf Average Area [cmA2] 80 mM NaCl, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.32 B 0.32 B
MAB 10 0.32 B 0.56 A 74
Table 120; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 121
Gene Id RGR of Mean(Leaf Average Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.39 B 0.39 B
MAB 13 0.41 B 0.57 A 49
MAB 15 0.46 A* 0.54 A 40
MAB 17 0.46 A* 0.50 A* 30
Table 121; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
129
Table 122 depicts analyses of RGR of Leaf Average Area [cmA2] in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 122
Gene Id RGR of Mean(Leaf Average Area [cmA2] 80 mM NaCl, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.28 B 0.28 B
MAB2 0.41 B 0.80 A 184
MAB43 0.46 B 0.83 A 195
MAB44 0.40 B 0.73 A 160
Table 122; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 123 depicts analyses of Plot Dry weight (DW) in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 123
Gene Id Dry Weight 80 mM Nat [g] :i
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.00 B 4.00 B
ΜΑΒΙ 4.92 A 6.40 A 60
MAB 134 4.35 B 5.35 A 34
MAB15 4.42 B 5.57 A 39
MAB18 4.52 B 5.35 A 34
MAB3GA 4.53 B 5.47 A 37
Table 123; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 124-126 depict analyses of 1000 Seeds Weight in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not
130 connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 124
Gene Id 1000 Seeds Weight [g] 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB 14 0.02 B 0.03 A 32
MAB19 0.02 B 0.03 A 27
MAB2 0.02 B 0.03 A 24
MAB6 0.03 A 0.03 A 53
Table 124; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 125
Gene Id 1000 Seeds Weight [g] 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB20 0.02 A* 0.02 A 17
MAB25 0.02 B 0.02 A 20
MAB6 0.02 A* 0.02 A 21
MAB7 0.02 B 0.02 A 21
MAB9 0.02 B 0.02 A 19
Table 125; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 126
1000 Seeds Weight [g] 80 mM NaCl
LSM % improvement of best event LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB 100 0.02 B 0.02 A 28
MAB 134 0.02 B 0.02 A 26
MAB 17 0.02 B 0.02 A 23
MAB 18 0.02 B 0.02 A 17
MAB32 0.02 B 0.02 A 13
MAB4 0.02 B 0.02 A 19
MAB46 0.02 B 0.02 A 18
MAB99 0.02 B 0.02 A 15
Table 126; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
131
Tables 127-129 depict analyses of Seed Yield per Plant in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 127
Gene Id Seed Yield per Plant [g] 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.07 B 0.07 B
MAB44 0.11 B 0.22 A 210
MAB50 0.11 B 0.19 A 170
Table 127; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 128
Gene Id Seed Yield per Plant [g] 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.09 B 0.09 B
MAB6 0.11 A* 0.21 A 142
MAB9 0.09 B 0.14 A 59
Table 128; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 129
Gene Id Seed Yield per Plant [g] 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.14 B 0.14 B
ΜΑΒΙ 0.19 A 0.33 A 139
MAB 100 0.17 B 0.24 A 79
Table 129; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 130 depicts analyses of Harvest Index in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not
132 connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 130
Gene Id Harvest Index 80 mM NaCl
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.11 B 0.11 B
MAB25 0.16 B 0.26 A 139
MAB44 0.20 A* 0.30 A 174
MAB7 0.12 B 0.29 A 172
Table 130; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 131-140 depict analyses of Rosette Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 131
Gene Id Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.37 B 1.37 B
ΜΑΒΙ 1.43 B 1.80 A 31
MAB9 1.32 B 1.74 A 27
Table 131; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 132
Gene Id Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.73 B 4.73 B
ΜΑΒΙ 4.95 B 6.45 A 36
Table 132; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
133
Table 133
Gene Id Rosette Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 8.45 B 8.45 B
ΜΑΒΙ 8.87 B 11.11 A 31
Table 133; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 134
Gene Id Rosette Area [cmA2] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.65 B 1.65 B
ΜΑΒΙ 2.09 A 2.27 A 37
MAB36 1.65 B 2.58 A 56
MAB7 1.83 B 2.81 A 70
Table 134; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 135
Gene Id Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.93 B 2.93 B
ΜΑΒΙ 3.60 A* 3.78 A* 29
MAB36 2.91 B 4.55 A 55
MAB7 3.14 B 4.69 A 60
Table 135; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 136
Gene Id Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 7.73 B 7.73 B
ΜΑΒΙ 9.77 A 10.58 A 37
MAB36 8.05 B 12.12 A 57
MAB7 8.69 B 12.82 A 66
Table 136; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
134
Table 137
2017228711 15 Sep 2017
Gene Id Rosette Area [cmA2] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.55 B 0.55 B
ΜΑΒΙ 0.58 B 0.81 A 47
MAB 100 0.60 B 0.74 A 34
MAB 15 0.65 A* 0.90 A 64
MAB 17 0.55 B 0.85 A 55
Table 137; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 138
Gene Id Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.03 B 1.03 B
ΜΑΒΙ 1.17 B 1.54 A 49
MAB 100 1.18 B 1.46 A 42
MAB15 1.23 A 1.67 A 62
MAB17 1.01 B 1.59 A 54
Table 138; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 139
Gene Id Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.09 B 2.09 B
ΜΑΒΙ 2.46 B 3.43 A 64
MAB 100 2.29 B 2.81 A 34
MAB 15 2.60 A 3.63 A 73
MAB 17 2.06 B 3.35 A 60
Table 139; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 140
Gene Id Rosette Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 4.81 B 4.81 B
ΜΑΒΙ 5.57 A* 8.29 A 72
MAB 15 5.72 A 8.05 A 67
MAB 17 4.78 B 7.50 A 56
135
Table 140; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Tables 141-148 depict analyses of Rosette Diameter in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 141
Gene Id Rosette Diameter [cm]
Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 3.52 B 3.52 B
ΜΑΒΙ 3.58 B 4.17 A 18
Table 141; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 142
Gene Id Rosette Diameter [cm] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.28 B 2.28 B
MAB36 2.23 B 2.91 A 28
MAB7 2.47 B 3.11 A 36
Table 142; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 143
Gene Id Rosette Diameter [cm] Normal conditions 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.99 B 2.99 B
MAB7 3.24 B 4.08 A 36
Table 143; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
136
Table 144
Gene Id Rosette Diameter [cm] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 5.00 B 5.00 B
ΜΑΒΙ 5.65 A* 5.87 A* 17
MAB7 5.06 B 6.32 A 26
Table 144; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 145
Gene Id Rosette Diameter [cm] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.30 B 1.30 B
MAB 15 1.48 A 1.69 A 30
MAB 17 1.33 B 1.60 A 23
Table 145; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 146
Gene Id Rosette Diameter [cm] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.87 B 1.87 B
ΜΑΒΙ 1.86 B 2.21 A 18
MAB 15 1.96 B 2.29 A 22
MAB 17 1.78 B 2.26 A 21
Table 146; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 147
Gene Id Rosette Diameter [cm] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 2.49 B
ΜΑΒΙ 2.60 B 3.14 A 26
MAB 15 2.64 B 3.17 A 27
MAB 17 2.39 B 3.09 A 24
Table 147; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
137
Table 148
2017228711 15 Sep 2017
Gene Id Rosette Diameter [cm] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 3.49 B 3.49 B
ΜΑΒΙ 3.88 A* 4.81 A 38
MAB 15 3.78 B 4.52 A 29
MAB 17 3.53 B 4.45 A 27
Table 148; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 149-157 depict analyses of Leaf Average Area in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 149
Gene Id Leaf Average Area [cmA2]
Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.17 B 0.17 B
ΜΑΒΙ 0.17 B 0.21 A 27
Table 149; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 150
Gene Id Leaf Average Area [cmA2]
Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.51 B 0.51 B
ΜΑΒΙ 0.52 B 0.69 A 35
Table 150; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 151
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.19 B 0.19 B
ΜΑΒΙ 0.25 A 0.27 A 38
MAB36 0.20 B 0.31 A 58
MAB7 0.23 A* 0.33 A 67
138
Table 151; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
2017228711 15 Sep 2017
Table 152
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.32 B 0.32 B
ΜΑΒΙ 0.38 B 0.43 A 34
MAB36 0.32 B 0.46 A 43
MAB7 0.33 B 0.47 A 45
Table 152; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 153
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.69 B 0.69 B
MAB36 0.69 B 0.93 A 36
MAB7 0.79 B 1.17 A 71
Table 153; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 154
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.11 B 0.11 B
ΜΑΒΙ 0.12 B 0.15 A 28
MAB 15 0.13 B 0.17 A 53
MAB 17 0.11 B 0.15 A 34
Table 154; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 155
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.16 B 0.16 B
ΜΑΒΙ 0.17 B 0.21 A 26
MAB 100 0.18 B 0.21 A 30
MAB 15 0.18 A* 0.23 A 39
MAB 17 0.16 B 0.22 A 35
139
Table 155; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
2017228711 15 Sep 2017
Table 156
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.24 B 0.24 B
ΜΑΒΙ 0.28 A* 0.37 A 50
MAB 15 0.29 A* 0.37 A 53
MAB 17 0.25 B 0.34 A 40
Table 156; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 157
Gene Id Leaf Average Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.54 B 0.54 B
ΜΑΒΙ 0.57 B 0.80 A 49
MAB 15 0.59 B 0.78 A 45
MAB 17 0.51 B 0.74 A 37
Table 157; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 158-166 depict analyses of RGR Rosette Area [cmA2] of plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 158
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 1.73 B 1.73 B
MAB20 2.18 B 3.62 A 109
MAB43 2.04 B 3.80 A 119
MAB50 2.25 B 3.81 A 120
Table 158; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
140
Table 159
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.48 B 0.48 B
MAB2 0.58 A* 0.70 A 45
MAB43 0.62 A 0.75 A 56
MAB6 0.52 B 0.72 A 50
Table 159; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 160
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.84 B 0.84 B
MAB50 0.87 B 0.99 A 18
MAB6 0.87 B 1.06 A 26
Table 160; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 161
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.39 B 0.39 B
MAB 10 0.44 B 0.54 A 37
MAB36 0.45 B 0.51 A 30
MAB50 0.45 A* 0.53 A 35
MAB6 0.44 B 0.60 A 51
MAB7 0.43 B 0.50 A 27
Table 161; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 162
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.39 B 0.39 B
MAB20 0.38 B 0.50 A 28
MAB25 0.39 B 0.53 A 38
Table 162; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
141
Table 163
2017228711 15 Sep 2017
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.55 B 0.55 B
MAB 10 0.64 A* 0.71 A* 30
MAB2 0.63 A* 0.70 A 28
MAB20 0.63 A* 0.67 A* 21
MAB25 0.64 A 0.73 A 32
MAB44 0.65 A 0.77 A 41
MAB50 0.70 A 0.83 A 51
MAB6 0.63 A* 0.81 A 48
MAB7 0.61 B 0.73 A 34
MAB9 0.60 B 0.69 A 26
Table 163; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 164
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.45 B 0.45 B
MAB13 0.63 A 0.68 A* 49
MAB32 0.50 B 0.74 A 64
MAB46 0.52 B 0.75 A 65
MAB 146 0.64 A 0.88 A 94
MAB99 0.52 B 0.73 A 61
Table 164; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 165
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.34 B 0.34 B
ΜΑΒΙ 0.36 B 0.45 A 31
MAB99 0.33 B 0.43 A 28
Table 165; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
142
Table 166
2017228711 15 Sep 2017
Gene Id RGR of Rosette Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.66 B 0.66 B
MAB13 0.73 B 0.81 A 23
MAB3 GA 0.70 B 0.85 A 29
MAB32 0.70 B 0.86 A 31
MAB99 0.68 B 0.82 A 25
Table 166; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 167-175 depict analyses of RGR of Rosette Diameter in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 167
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.43 B 0.43 B
MAB50 0.70 A* 1.50 A 251
MAB6 0.45 B 1.21 A 183
Table 167; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 168
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.16 B 0.16 B
MAB 10 0.19 A* 0.21 A* 28
MAB19 0.20 A 0.23 A 45
MAB36 0.18 B 0.21 A 32
MAB50 0.17 B 0.23 A 42
MAB6 0.18 B 0.25 A 57
MAB7 0.18 B 0.24 A 52
Table 168; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
143
Table 169
2017228711 15 Sep 2017
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.25 B 0.25 B
MAB 10 0.28 A 0.30 A 19
MAB 14 0.27 B 0.31 A 23
MAB19 0.28 A 0.32 A 29
MAB2 0.27 B 0.30 A 21
MAB20 0.27 B 0.29 A 18
MAB36 0.27 A* 0.32 A 28
MAB43 0.25 B 0.26 B 5
MAB44 0.26 B 0.30 A 21
MAB50 0.27 B 0.30 A 21
MAB7 0.28 A* 0.29 A 17
MAB9 0.27 A* 0.30 A 20
Table 169; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 170
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.17 B 0.17 B
MAB19 0.19 A* 0.23 A 31
MAB2 0.20 A 0.23 A 32
MAB20 0.19 A 0.23 A 33
MAB43 0.19 B 0.21 A 24
MAB44 0.18 B 0.22 A 25
MAB50 0.20 A 0.23 A 32
MAB6 0.19 A* 0.24 A 42
MAB9 0.18 B 0.21 A 25
Table 170; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 171
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 5from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.16 B 0.16 B
MAB50 0.19 B 0.22 A 42
MAB6 0.15 B 0.24 A 49
Table 171; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
144
Table 172
2017228711 15 Sep 2017
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.22 B 0.22 B
MAB2 0.26 A* 0.28 A 27
MAB20 0.26 B 0.30 A 33
MAB25 0.26 A* 0.29 A* 31
MAB43 0.24 B 0.29 A 29
MAB44 0.25 B 0.29 A 31
Table 172; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
Table 173
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 3 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.29 B 0.29 B
MAB 100 0.37 A* 0.51 a 74
MAB13 0.38 A 0.58 A 95
MAB15 0.36 A 0.45 A 54
MAB18 0.36 A* 0.38 A* 28
MAB3 GA 0.43 A 0.60 A 105
MAB35 0.39 A 0.44 A 50
MAB46 0.31 B 0.49 A 65
MAB 146 0.35 A 0.44 A 50
Table 173; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 174
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.11 B 0.11 B
ΜΑΒΙ 0.13 A* 0.16 A 49
MAB13 0.13 A* 0.16 A 41
MAB18 0.14 A 0.16 A 45
MAB32 0.13 B 0.15 A 39
MAB 146 0.16 A 0.19 A 72
MAB99 0.12 B 0.15 A 40
Table 174; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
145
Table 175
2017228711 15 Sep 2017
Gene Id RGR of Rosette Diameter [cm]) Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.20 B 0.20 B
ΜΑΒΙ 0.25 A 0.27 A 30
MAB17 0.24 A 0.26 A* 25
MAB18 0.25 A 0.31 A 51
MAB35 0.25 A 0.28 A 36
MAB 146 0.25 A 0.28 A 36
MAB99 0.24 A 0.29 A 44
Table 175; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 176-178 depict analyses of RGR of Leaf Average Area [cmA2] in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 176
Gene Id RGR of Mean(Leaf Average Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.34 B 0.34 B
MAB 10 0.35 B 0.52 A 56
MAB36 0.40 B 0.52 A 55
MAB7 0.37 B 0.50 A 49
Table 176; LSM = Least square mean; % improvement = compare to control (GUI). The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 177
Gene Id RGR of Mean(Leaf Average Area [cmA2] Normal conditions, Day 10 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.38 B 0.38 B
MAB 10 0.47 A 0.51 A* 35
MAB2 0.41 B 0.49 A 29
MAB25 0.43 B 0.55 A 44
MAB50 0.47 A 0.53 A 41
MAB7 0.45 A* 0.50 A* 31
MAB9 0.43 B 0.54 A 41
Table 177; LSM = Least square mean; % improvement = compare to control (GUI). The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
146
Table 178
2017228711 15 Sep 2017
Gene Id RGR of Mean(Leaf Average Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.23 B 0.23 B
MAB13 0.34 A* 0.39 A* 70
MAB 146 0.35 A* 0.50 A 117
MAB99 0.26 B 0.44 A 89
Table 178; LSM = Least square mean; % improvement = compare to control (GUI). The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 179-180 depict analyses of RGR of Leaf Average Area [cmA2] in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 179
Gene Id RGR of Mean(Leaf Average Area [cmA2] Normal conditions, Day 5 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.31 B 0.31 B
MAB19 0.35 B 0.48 A 56
MAB43 0.39 B 0.52 A 70
MAB6 0.28 B 0.50 A 62
Table 179; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 180
Gene Id RGR of Mean(Leaf Average Area [cmA2] Normal conditions, Day 8 from planting
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.69 B 0.69 B
MAB 14 0.72 B 0.92 A 32
MAB6 0.69 B 0.96 A 38
Tables 181-182 depict analyses of Plot Dry weight (DW) in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events
Table 180; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
147 per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
2017228711 15 Sep 2017
Table 181
Gene Id Dry Weight [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 7.75 B 7.75 B
MAB36 10.37 A* 13.21 A 71
Table 181; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 182
Gene Id Dry Weight [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 5.23 B 5.23 B
ΜΑΒΙ 6.81 A 8.09 A 55
MAB 13 6.08 B 7.61 A 45
MAB 18 6.10 B 8.18 A 56
MAB99 6.51 A* 8.42 A 61
Table 182; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 183-185 depict analyses of 1000 Seeds Weight in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 183
Gene Id 1000 Seeds Weight [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB 19 0.02 B 0.03 A 23
MAB2 0.02 B 0.03 A 44
MAB20 0.02 A 0.04 A 71
MAB36 0.02 B 0.03 A 24
MAB50 0.02 B 0.03 A 32
MAB6 0.02 B 0.03 A 22
MAB9 0.02 A 0.02 A 19
148
Table 183; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
2017228711 15 Sep 2017
Table 184
Gene Id 1000 Seeds Weight [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB20 0.02 A* 0.02 A 17
MAB6 0.02 A* 0.02 A 21
Table 184; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Table 185
Gene Id 1000 Seeds Weight [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.02 B 0.02 B
MAB 100 0.02 B 0.02 A 23
MAB 17 0.02 A 0.03 A 33
MAB 18 0.02 B 0.02 A 18
MAB35 0.02 B 0.02 A 28
MAB46 0.02 A 0.02 A 21
MAB99 0.02 A 0.03 A 37
Table 185; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
Tables 186-187 depict analyses of Seed Yield per Plant in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 186
Gene Id Seed Yield per Plant [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.38 B 0.38 B
ΜΑΒΙ 0.50 B 0.61 A 61
MAB 10 0.46 B 0.59 A 53
MAB 14 0.50 A* 0.60 A 57
MAB36 0.52 A 0.68 A 77
MAB50 0.46 B 0.60 A 56
149
Table 186; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in
Table 3 above.
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Table 187
Gene Id Seed Yield per Plant [g] Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.32 B 0.32 B
ΜΑΒΙ 0.41 A* 0.49 A 53
MAB 13 0.43 A 0.55 A 69
MAB 18 0.39 B 0.49 A 53
MAB32 0.41 B 0.50 A 56
MAB35 0.41 A* 0.50 A 57
MAB99 0.41 A* 0.51 A 57
Table 187; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants is provided in Table 3 above.
Table 188 depicts analyses of Harvest Index in plants overexpressing the polynucleotides of the invention under the regulation of 6669 promoter. Each Table represents an independent experiment, using 4 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.
Table 188
Gene Id Harvest Index Normal conditions
LSM Significance LSM of Best event Significance % improvement of best event
GUI 0.48 B 0.48 B
MAB 17 0.46 B 0.62 A 28
Table 188; LSM = Least square mean; % improvement = compare to control (GUI); A meaning significant different at P < 0.05, A* meaning significant different at P < 0.1. The SEQ ID NOs. of the cloned genes (according to the Gene Id) which are exogenously expressed in the plants are provided in Table 3 above.
EXAMPLE 8
TRANSFORMA TION OF TOMA TO M82 PLANTS WITH PUTA TIVE ABST
GENES
For the tomato transformation, tomato M82 seeds were previously sterilized with Na-hipochloride 3 % + 2-3 drops of Tween 20 (Polysorbate 20). Seeds were washed 3 times with distilled sterile water. Seeds were then germinated in full strength Nitsch medium and germinated for 8 days 8 days in growth room at 25 °C in the dark. Plantlets were then cut with 2-4 cm stem and insert it into alO-cm Petri dishes that were
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150 filled with 30-40 ml of MS liquid medium. Cotyledons were then cut and used as explants and later transferred onto KCMS solidified medium with 100 μΜ acetosyringone in a 10-cm Petri dish. Explants were inoculated with A. tumefascience for 30-50 minutes. Explants were co-cultivated for 24 hours and transferred to regeneration media including Kanamycin as selection medium. The resistant regenerated plantlets were then transferred into a rooting medium for 10-14 days until the appearance of the roots.
EXAMPLE 9
GROWTH OF M82 TOMATO TRANSFORMED PLANTS AND PHENOTYPE CHARACTERIZA TIONS
Experimental Procedures
Producing transgenic tomato plants - Plants were transformed as described in Example 8, above. Following transformation, ΤΙ M82 tomato plants were grown until fruit set. T2 seeds have entered experiments to assess abiotic stress resistance.
Experimental Results
Assay 1 - Tomato field trial under regular and water deficient regimes - The tomato field trial was planned as a one source dripping irrigation (OSDI) system similar to a standard farmer field. Since water deficiency is applied in a relatively uniform manner, it allows measuring the effect of drought on small size populations of plants. The OSDI method was developed on the basis of the line source sprinklers irrigation system (Hanks et al. 1976 Soil Sei. Soc Am. J. 40 p. 426-429) with some significant modifications. Instead of sprinkler irrigation, dripping irrigation was used. In order to create a uniform and deep wet layer (at least 60 cm depth), and not the onion shape layer that is typically created by dripping irrigation, a low pressure compensating dripping irrigation system was used. This system enables to supply small amounts of water in a relatively long time frame. The drought stress field trial was performed in light soil, in an open field (net-house) near Rehovot, Israel. Between 4 to 5 events are been evaluated for each gene and the null segregating populations are used as negative controls. During the first three weeks all plants were grown in a nursery under normal irrigation conditions. After this period, plants were transplanted according to commercial growth protocol, maintaining a 30 cm distance between plants reaching a
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151 total density of 2,600 plants per 1000 sq. m (the recommended density in commercial growth). Each plant was transplanted near a water dripper and further subjected to two different treatments:
Optimal (100 %): optimal irrigation conditions (100 %). Irrigation was applied every 2 days as a standard recommended water supply. Standard recommended water supply is the amount applied by local commercial growers according to standard protocols.
Severe Stress (50 %): 50 % of the optimal amount of water irrigation was applied once a day (at same time as regular irrigation is applied)
All fertilizers were applied according to local standard protocols. Nitrogen was equally applied, as recommended, to all the treatments through the irrigation system. Each row, 193 cm wide, contained two dripping irrigation lines creating coverage of six drippers per 1 sq. m. The irrigation control was performed separately for each treatment. The experiment was structured in a four randomized block design, eight plants per plot. The different water regimes were initiated only four weeks three transplantation, when plants initiated the flowering stage. Water availability in the soil was recorded using tensiometers (used to determine matric water potential Ψιη which allows to evaluate the stress severeness).
Assay 2 - Tomato salt bath experiment - Transgenic tomato seeds are sown in trays containing growth denitrified media. Seedlings are germinated under nursery conditions. The experimental model used was 3 blocks random distributed, where 10 plants per events were sown in each block. At the stage of first true leaf, trays are transferred to different tanks containing growth solution of 300 mM NaCl. For normal treatment, a full Hoagland solution was applied. 5 events for each gene are evaluated while null segregating populations are used as negative controls. The experiment is performed for a period of 8 weeks, where parameters such as chlorophyll content (measured as SPAD units), plant biomass (FW and DW) are measured.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
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All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or 5 identification of any 5 reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Claims (20)

  1. CLAIMS:
    1. A method of increasing biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance of a plant, the method comprising over-expressing within the plant a polypeptide comprising an amino acid sequence at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 271, wherein when said abiotic stress is salinity stress then said tolerance to said salinity stress is an increase in said seed yield and/or an increase in said growth rate under salinity stress as compared to a native plant of the same species which is grown under the same growth conditions, thereby increasing the biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance of the plant, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency.
  2. 2. The method of claim 1, further comprising selecting plants over-expressing said polypeptide for an increased biomass, growth rate, seed yield, abiotic stress tolerance and/or nitrogen use efficiency as compared to a control plant of the same species which is grown under the same growth conditions, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency.
  3. 3. A method of growing a crop comprising growing a crop plant over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, wherein said crop plant is derived from parent plants over-expressing said polypeptide as compared to a native plant of the same species which is grown under the same growth conditions, and which have been selected for increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance of a plant, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency, and said crop plant over-expressing said polypeptide having said increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, wherein said tolerance to said salinity stress is an increase in said seed yield and/or an increase in said growth rate under said salinity stress as compared to a native plant of the same species which is grown under the same growth conditions, thereby growing the crop.
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  4. 4. The method of claim 3, wherein said growing of said crop plant over-expressing said polypeptide is performed under salinity conditions.
  5. 5. The method of claim 3, wherein said growing of said crop plant over-expressing said polypeptide is performed under nitrogen deficient conditions.
  6. 6. A method of selecting a plant having increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, the method comprising:
    (a) providing plants over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, (b) selecting said plants of step (a) for increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance, wherein the abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency, wherein said tolerance to said salinity stress is an increase in said seed yield and/or an increase in said growth rate under salinity stress as compared to a native plant of the same species which is grown under the same growth conditions, and (c) growing a crop of said plant selected in step (b), thereby selecting the plant having the increased biomass, growth rate, seed yield, nitrogen use efficiency and/or abiotic stress tolerance.
  7. 7. A method of increasing root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or increased seed yield of a plant as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of a control plant of the same species which is grown under the same growth conditions, comprising:
    (a) over-expressing within the plant a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, and (b) selecting from plants resultant of step (a) a plant exhibiting an increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said selecting is for a plant exhibiting said increased seed yield and/or said increased growth rate as compared to said control plant of the same species which is grown under the same growth conditions,
    155
    2017228711 11 Dec 2018 thereby increasing the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of the plant as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of the control plant of the same species which is grown under the same growth conditions.
  8. 8. The method of claim 7, wherein said selecting in step (b) is performed under normal conditions.
  9. 9. The method of claim 7, wherein said selecting in step (b) is performed under an abiotic stress, wherein said abiotic stress is selected from the group consisting of salinity stress and nitrogen deficiency.
  10. 10. The method of claim 7, wherein said selecting said plant exhibiting said increased root length, said increased growth rate of rosette area, said increased growth rate of rosette diameter and/or said increased seed yield is performed under normal conditions.
  11. 11. The method of claim 7, wherein said selecting said plant exhibiting said increased root coverage is performed under nitrogen deficient conditions.
  12. 12. The method of claim 7, wherein said selecting said plant exhibiting said increased growth rate of rosette area, said increased growth rate of rosette diameter, and/or said increased seed yield is performed under a salinity stress.
  13. 13. A method of producing a crop comprising growing a crop plant over-expressing a polypeptide comprising an amino acid sequence at least 80 % identical to the polypeptide set forth in SEQ ID NO: 271, wherein said crop plant is derived from parent plants selected for increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to the root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield of a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said parent plants are selected for said increased seed yield and/or said increased growth rate as compared to said control plant of the same species which is grown under the same growth conditions, and said crop plant having said increased root length, root
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    2017228711 11 Dec 2018 coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield, thereby producing the crop.
  14. 14. A method of selecting a plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, the method comprising:
    (a) providing plants transformed with a nucleic acid construct comprising a polynucleotide comprising a nucleic acid sequence encoding a polypeptide, wherein said polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 271, and a heterologous promoter for directing transcription of said nucleic acid sequence in a plant cell, and;
    (b) selecting from said plants a plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to a control plant of the same species which is grown under the same growth conditions, wherein when said growth conditions comprise salinity stress then said selecting is for a plant having said increased seed yield and/or said increased growth rate as compared to said control plant of the same species which is grown under the same growth conditions, thereby selecting the plant having increased root length, root coverage, growth rate of rosette area, growth rate of rosette diameter, and/or seed yield as compared to the control plant of the same species which is grown under the same growth conditions.
  15. 15. The method of any one of claims 1 to 14, wherein said amino acid sequence is at least 85 % identical to the amino acid sequence set forth in SEQ ID NO: 271.
  16. 16. The method of any one of claims 1 to 14, wherein said amino acid sequence is at least 90 % identical to the amino acid sequence set forth in SEQ ID NO: 271.
  17. 17. The method of any one of claims 1 to 14, wherein said amino acid sequence is at least 95 % identical to the amino acid sequence set forth in SEQ ID NO: 271.
  18. 18. The method of any one of claims 1 to 14, wherein said amino acid sequence is set forth in SEQ ID NO: 271.
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  19. 19. The method of claim 14, wherein said polynucleotide is selected from the group consisting of SEQ ID NOs: 1559 and 81.
  20. 20. The method of any one of claims 1, 7 and 15 to 18, further comprising growing the plant over-expressing said polypeptide under the abiotic stress.
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US20050054831A1 (en) * 2001-02-02 2005-03-10 In-Hwan Hwang Osmotic stress-inducible protein functioning as a negative regulator in osmotic stress signaling pathway of plants
US20060021088A1 (en) * 2002-10-18 2006-01-26 Dirk Inze Identification of novel e2f target genes and use thereof
US20070039067A1 (en) * 2004-09-30 2007-02-15 Ceres, Inc. Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics

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